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Abstract:

The invention provides a human PRCP which is associated with the
cardiovascular diseases, hematological diseases, neurological diseases
and cancer. The invention also provides assays for the identification of
compounds useful in the treatment or prevention of cardiovascular
diseases, hematological diseases, neurological diseases and cancer. The
invention also features compounds which bind to and/or activate or
inhibit the activity of PRCP as well as pharmaceutical compositions
comprising such compounds.

Claims:

1. A method of screening for therapeutic agents useful in the treatment of
cardiovascular diseases, hematological diseases, neurological diseases or
cancer in a mammal comprising the steps of:contacting a test compound
with a PRCP polypeptide, anddetect binding of said test compound to said
PRCP polypeptide;ordetermining the activity of a PRCP polypeptide at a
certain concentration of a test compound or in the absence of said test
compound, anddetermining the activity of said polypeptide at a different
concentration of said test compound;ordetermining the activity of a PRCP
polypeptide at a certain concentration of a test compound, anddetermining
the activity of a PRCP polypeptide at the presence of a compound known to
be a regulator of a PRCP polypeptide.

2-3. (canceled)

4. The method of claim 1, wherein the step of contacting is in or at the
surface of a cell.

5. The method of claim 1, wherein the cell is in vitro.

6. The method of claim 1, wherein the step of contacting is in a cell-free
system.

7. The method of claim 1, wherein the polypeptide is coupled to a
detectable label.

8. The method of claim 1, wherein the compound is coupled to a detectable
label.

9. The method of claim 1, wherein the test compound displaces a ligand
which is first bound to the polypeptide.

10. The method of claim 1, wherein the polypeptide is attached to a solid
support.

11. The method of claim 1, wherein the compound is attached to a solid
support.

12. A method of screening for therapeutic agents useful in the treatment
of cardiovascular diseases, hematological diseases, neurological diseases
or cancer in a mammal comprising the steps of:contacting a test compound
with a PRCP polynucleotide,detect binding of said test compound to said
PRCP polynucleotide;ordetermining the amount of a PRCP polynucleotide in
a sample taken from said mammal,determining the amount of PRCP
polynucleotide in healthy and/or diseased mammals.

13. The method of claim 12 wherein the nucleic acid molecule is RNA.

14. The method of claim 12 wherein the contacting step is in or at the
surface of a cell.

15. The method of claim 12 wherein the contacting step is in a cell-free
system.

16. The method of claim 12 wherein polynucleotide is coupled to a
detectable label.

17. The method of claim 12 wherein the test compound is coupled to a
detectable label.

18-26. (canceled)

Description:

TECHNICAL FIELD OF THE INVENTION

[0001]The present invention is in the field of molecular biology, more
particularly, the present invention relates to nucleic acid sequences and
amino acid sequences of a human PRCP and its regulation for the treatment
of cardiovascular diseases, hematological diseases, neurological diseases
and cancer in mammals.

BACKGROUND OF THE INVENTION

[0002]Proteases play a role in carefully controlled processes, such as
blood coagulation, fibrinolysis, complement activation, fertilization,
and hormone production. These enzymes are also used in a variety of
diagnostic, therapeutic, and industrial contexts. PRCP is a member of the
group of protease enzymes [Watson et al. (1997); Tan et al. (1993);
EP1498424; WO 04/072265].

[0003]Proteases were recognized very early in the history of biochemistry.
In the nineteenth century, one primary focus of research was on digestive
proteases, like pepsin and trypsin. Proteases belong systematically to
the C--N Hydrolases. More specifically, proteases catalyze the hydrolytic
cleavage of a peptide bond and are therefore called peptidases as well.

[0004]Proteases can be classified according to several criteria, e.g. by
localisation. Digestive proteases are located in the gastro-intestinal
tract. These proteases are responsible for the digestion of food
proteins.

[0005]Peptidases located extracellularly in the blood or other
extracellular compartments of the body play often regulatory roles in
processes like for example blood clotting, fibrinolysis, or activation of
complement constituents.

[0006]Intracellularly located proteases exhibit a wide variety of roles.
They are found in compartments like the ER, the Golgi apparatus, or the
lysosomes. Their functions include for example activation of peptide
hormons, ubiquitin mediated proteolysis, among others.

[0007]Proteases are most commonly classified according to their mechanism
of action, or to specific active groups that are present in the active
center. The following groups can be distinguished:

[0009]Serine proteases exhibit a serine in the catalytic site which forms
a covalent ester intermediate during the catalytic reaction pathway by a
nucleophilic attack on the carboxy carbon of the peptide bond. In the
active site of serine proteases a catalytic triad comprised of an
aspartate, a histidine and the above mentioned serine is found. This
triad functions in the reaction mechanism as a charge relay system.

[0010]To the large family of serine protease belong, for example, the
digestive enzymes trypsin and chymotrypsin, components of the complement
cascade, enzymes involved in the blood clotting cascade, as well as
enzymes that function in degradation, rebuilding and maintenace of
constituents of the extracellular matrix.

[0011]One feature of the serine protease family is the broad range of
substrate specificity. Members of the trypase subgroup cleave after
arginine or lysine, chymases after phenylalanine or leucine, aspases
after aspartate, metases after methionine and serases after serine.

Cysteine Proteases

[0012]During the catalytic reaction of cysteine proteases a covalent
thioester intermediate is formed by a nucleophilic attack of the cysteine
on the caboxy carbon of the peptide bond. Similar to the serine
peptidases a catalytic triad comprised of the cysteine, a histidine and
an asparagine is found which functions as a charge relay system to
facilitate the formation of the thioester intermediate.

[0013]Members of the Cysteine protease family have roles in many different
cellular processes, e.g. processing of precursers or intracellular
degradation. Examples for cysteine proteases include lysosomal
cathepsines, and cytosolic calpains.

Aspartyl- or Acidic Peptidase

[0014]The catalytic site of aspartyl proteases is composed of two
aspartate residues. At the pH optimum of aspartyl proteases (2-3) one of
the aspartyl carboxy groups is ionized and the other is neutral, which is
important for the catalytic reaction to occur. Examples for aspartyl
proteases are gastric pepsins A and C, chymosin, as well as mammalian
renin.

Metallo-peptidases

[0015]Metallo-peptidases are proteases, whose proteolytic activity depends
on the presence of divalent cations in the active center. Examples of
members of this class are carboxypeptidase A, which represents a
pancreatic digestive enzyme, the Angiotension Converting Enzymes (ACE),
which are responsible for the conversion of angiotensin I to angiotensin
II, or the Extracellular Matrix Metalloproteases.

[0016]In summary, a huge number of proteases play a central role in
several important cellular and intracellular processes. Furthermore, the
value as pharmaceutical targets has been proven for several proteases.
For example, the protease encoded by the HIV genome is used as a target
for drugs for the treatment of HIV infections, the proteasom complex has
been discovered as an anti-cancer target, or Cys-proteases have been
implemented as drug targets for inflammatory disorders. Selective
inhibitors have been developed as therapeutic agents for diseases such as
HIV. Thus, the identification of further disease implications of protease
species and their splice variants may lead to the development of specific
inhibitors or modulators, or suggest new utilities for known compounds
affecting proteases. That in turn will provide additional pharmacological
approaches to treat diseases and conditions in which protease activities
are involved. This diseases may include, but are not limited to,
infections such as bacterial, fungal, protozoan, and viral infections,
particularly those caused by HIV viruses, cancers, allergies including
asthma, cardiovascular diseases including acute heart failure,
hypotension, hypertension, angina pectoris, myocardial infarction,
hematological diseases, genito-urinary diseases including urinary
incontinence and benign prostate hyperplasia, osteoporosis, peripheral
and central nervous system disorders including pain, Alzheimer's disease
and Parkinson's disease, respiratory diseases, metabolic diseases,
inflammatory diseases, gastro-enterological diseases, diseases of the
endocrine system, dermatological diseases, diseases of muscles or the
skeleton, immunological diseases, developmental diseases or diseases of
the reproductive system.

TaqMan-Technology/expression Profiling

[0017]TaqMan is a recently developed technique, in which the release of a
fluorescent reporter dye from a hybridisation probe in real-time during a
polymerase chain reaction (PCR) is proportional to the accumulation of
the PCR product. Quantification is based on the early, linear part of the
reaction, and by determining the threshold cycle (CT), at which
fluorescence above background is first detected.

[0018]Gene expression technologies may be useful in several areas of drug
discovery and development, such as target identification, lead
optimization, and identification of mechanisms of action. The TaqMan
technology can be used to identify genes with selective tissue
distribution which may serve as indirect clues about disease-causing
genes or drug targets. Furthermore, the TaqMan technology can be used to
compare differences between expression profiles of normal tissue and
diseased tissue. Expression profiling has been used in identifying genes,
which are up- or downregulated in a variety of diseases. An interesting
application of expression profiling is temporal monitoring of changes in
gene expression during disease progression and drug treatment or in
patients versus healthy individuals. The premise in this approach is that
changes in pattern of gene expression in response to physiological or
environmental stimuli (e.g., drugs) may serve as indirect clues about
disease-causing genes or drug targets. Moreover, the effects of drugs
with established efficacy on global gene expression patterns may provide
a guidepost, or a genetic signature, against which a new drug candidate
can be compared.

PRCP

[0019]The nucleotide sequence of PRCP is accessible in the databases by
the accession number L13977 and is given in SEQ ID NO:1. The amino acid
sequence of PRCP depicted in SEQ ID NO:2.

[0020]Prolylcarboxypeptidase (peptidyl prolylamino acid hydrolase; cleaves
C-terminal amino acids linked to a penultimate proline, if proline has a
protected amino group or is part of a peptide chain. As a lysosomal
enzyme, it has a sharp acid pH optimum with shorter substrates, but with
longer peptides, 20 to 90% of the activity is retained at neutral pH. The
enzyme was discovered when a swine kidney extract unexpectedly cleaved
bradykinin. Because angiotensins II and III have the same C terminus,
-pro-phe-OH, and are substrates for the enzyme, prolylcarboxypeptidase
was named angiotensinase C. The protein comprises 451 amino acids. It was
detected in a variety of human cells, including white blood cells,
fibroblasts, and endothelial cells, and organs such as kidney and lung,
and it is excreted in urine. Tan et al. [Tan et al. (1993)] reported the
purification and partial protein sequencing of PCP and the cloning and
sequencing of its cDNA.

[0021]Because of the role of angiotensin II in the renin-angiotensin
system which regulates blood pressure and electrolyte balance, the PCP
gene is a candidate gene for essential hypertension. Watson et al.
[Watson et al. (1997)] mapped the gene to chromosome 11 by PCR analysis
of a somatic cell hybrid mapping panel and refined the localization by
screening a chromosome 11-specific YAC library using PCR-based methods.
By fluorescence in situ hybridization, they localized the gene to 11q14.
They also determined its position in relation to a chromosome 11
radiation hybrid map

[0022]PRCP is published, but not limited to, in patents EP1498424 and
WO2004072265.

SUMMARY OF THE INVENTION

[0023]The invention relates to novel disease associations of PRCP
polypeptides and polynucleotides. The invention also relates to novel
methods of screening for therapeutic agents for the treatment of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal. The invention also relates to pharmaceutical
compositions for the treatment of cardiovascular diseases, hematological
diseases, neurological diseases and cancer in a mammal comprising a PRCP
polypeptide, a PRCP polynucleotide, or regulators of PRCP or modulators
of PRCP activity. The invention further comprises methods of diagnosing
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal.

[0026]FIG. 3 shows the nucleotide sequence of a primer useful for the
invention (SEQ ID NO:3).

[0027]FIG. 4 shows the nucleotide sequence of a primer useful for the
invention (SEQ ID NO:4).

[0028]FIG. 5 shows a nucleotide sequence useful as a probe to detect
polynucleotides of the invention (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

[0029]An "oligonucleotide" is a stretch of nucleotide residues which has a
sufficient number of bases to be used as an oligomer, amplimer or probe
in a polymerase chain reaction (PCR). Oligonucleotides are prepared from
genomic or cDNA sequence and are used to amplify, reveal, or confirm the
presence of a similar DNA or RNA in a particular cell or tissue.
Oligonucleotides or oligomers comprise portions of a DNA sequence having
at least about 10 nucleotides and as many as about 35 nucleotides,
preferably about 25 nucleotides.

[0030]"Probes" may be derived from naturally occurring or recombinant
single- or double-stranded nucleic acids or may be chemically
synthesized. They are useful in detecting the presence of identical or
similar sequences. Such probes may be labeled with reporter molecules
using nick translation, Klenow fill-in reaction, PCR or other methods
well known in the art. Nucleic acid probes may be used in southern,
northern or in situ hybridizations to determine whether DNA or RNA
encoding a certain protein is present in a cell type, tissue, or organ.

[0031]A "fragment of a polynucleotide" is a nucleic acid that comprises
all or any part of a given nucleotide molecule, the fragment having fewer
nucleotides than about 6 kb, preferably fewer than about 1 kb.

[0032]"Reporter molecules" are radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents which associate with a particular
nucleotide or amino acid sequence, thereby establishing the presence of a
certain sequence, or allowing for the quantification of a certain
sequence.

[0033]"Chimeric" molecules may be constructed by introducing all or part
of the nucleotide sequence of this invention into a vector containing
additional nucleic acid sequence which might be expected to change any
one or several of the following PRCP characteristics: cellular location,
distribution, ligand-binding affinities, interchain affinities,
degradation/turnover rate, signaling, etc.

[0034]"Active", with respect to a PRCP polypeptide, refers to those forms,
fragments, or domains of a PRCP polypeptide which retain the biological
and/or antigenic activity of a PRCP polypeptide.

[0035]"Naturally occurring PRCP polypeptide" refers to a polypeptide
produced by cells which have not been genetically engineered and
specifically contemplates various polypeptides arising from
post-translational modifications of the polypeptide including but not
limited to acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation.

[0036]"Derivative" refers to polypeptides which have been chemically
modified by techniques such as ubiquitination, labeling (see above),
pegylation (derivatization with polyethylene glycol), and chemical
insertion or substitution of amino acids such as ornithine which do not
normally occur in human proteins.

[0037]"Conservative amino acid substitutions" result from replacing one
amino acid with another having similar structural and/or chemical
properties, such as the replacement of a leucine with an isoleucine or
valine, an aspartate with a glutamate, or a threonine with a serine.

[0038]"Insertions" or "deletions" are typically in the range of about 1 to
5 amino acids. The variation allowed may be experimentally determined by
producing the peptide synthetically while systematically making
insertions, deletions, or substitutions of nucleotides in the sequence
using recombinant DNA techniques.

[0039]A "signal sequence" or "leader sequence" can be used, when desired,
to direct the polypeptide through a membrane of a cell. Such a sequence
may be naturally present on the polypeptides of the present invention or
provided from heterologous sources by recombinant DNA techniques.

[0040]An "oligopeptide" is a short stretch of amino acid residues and may
be expressed from an oligonucleotide. Oligopeptides comprise a stretch of
amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15,
25 amino acids, typically of at least 9 to 13 amino acids, and of
sufficient length to display biological and/or antigenic activity.

[0041]"Inhibitor" is any substance which retards or prevents a chemical or
physiological reaction or response. Common inhibitors include but are not
limited to antisense molecules, antibodies, and antagonists.

[0042]"Standard expression" is a quantitative or qualitative measurement
for comparison. It is based on a statistically appropriate number of
normal samples and is created to use as a basis of comparison when
performing diagnostic assays, running clinical trials, or following
patient treatment profiles.

[0044]A "PRCP polynucleotide", within the meaning of the invention, shall
be understood as being a nucleic acid molecule selected from a group
consisting of [0045](i) nucleic acid molecules encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, [0046](ii) nucleic
acid molecules comprising the sequence of SEQ ID NO: 1, [0047](iii)
nucleic acid molecules having the sequence of SEQ ID NO: 1, [0048](iv)
nucleic acid molecules which hybridizes under stringent conditions to the
complementary strand of a nucleic acid molecule of (i), (ii), or (iii);
and [0049](v) nucleic acid molecules the sequence of which differs from
the sequence of a nucleic acid molecule of (iii) due to the degeneracy of
the genetic code;wherein the polypeptide encoded by said nucleic acid
molecule has PRCP activity.

[0050]A "PRCP polypeptide", within the meaning of the invention, shall be
understood as being a polypeptide selected from a group consisting of
[0051](i) polypeptides having the sequence of SEQ ID NO: 2, [0052](ii)
polypeptides comprising the sequence of SEQ ID NO: 2, [0053](iii)
polypeptides encoded by PRCP polynucleotides; and [0054](iv) polypeptides
which show at least 99%, 98%, 95%, 90%, or 80% homology with a
polypeptide of (i), (ii), or (iii);wherein said polypeptide has PRCP
activity.

[0055]The nucleotide sequences encoding a PRCP (or their complement) have
numerous applications in techniques known to those skilled in the art of
molecular biology. These techniques include use as hybridization probes,
use in the construction of oligomers for PCR, use for chromosome and gene
mapping, use in the recombinant production of PRCP, and use in generation
of antisense DNA or RNA, their chemical analogs and the like. Uses of
nucleotides encoding a PRCP disclosed herein are exemplary of known
techniques and are not intended to limit their use in any technique known
to a person of ordinary skill in the art. Furthermore, the nucleotide
sequences disclosed herein may be used in molecular biology techniques
that have not yet been developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known, e.g., the
triplet genetic code, specific base pair interactions, etc.

[0056]It will be appreciated by those skilled in the art that as a result
of the degeneracy of the genetic code, a multitude of PRCP--encoding
nucleotide sequences may be produced. Some of these will only bear
minimal homology to the nucleotide sequence of the known and naturally
occurring PRCP. The invention has specifically contemplated each and
every possible variation of nucleotide sequence that could be made by
selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet genetic
code as applied to the nucleotide sequence of naturally occurring PRCP,
and all such variations are to be considered as being specifically
disclosed.

[0057]Although the nucleotide sequences which encode a PRCP, its
derivatives or its variants are preferably capable of hybridizing to the
nucleotide sequence of the naturally occurring PRCP polynucleotide under
stringent conditions, it may be advantageous to produce nucleotide
sequences encoding PRCP polypeptides or its derivatives possessing a
substantially different codon usage. Codons can be selected to increase
the rate at which expression of the peptide occurs in a particular
prokaryotic or eukaryotic expression host in accordance with the
frequency with which particular codons are utilized by the host. Other
reasons for substantially altering the nucleotide sequence encoding a
PRCP polypeptide and/or its derivatives without altering the encoded
amino acid sequence include the production of RNA transcripts having more
desirable properties, such as a greater half-life, than transcripts
produced from the naturally occurring sequence.

[0058]Nucleotide sequences encoding a PRCP polypeptide may be joined to a
variety of other nucleotide sequences by means of well established
recombinant DNA techniques. Useful nucleotide sequences for joining to
PRCP polynucleotides include an assortment of cloning vectors such as
plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
Vectors of interest include expression vectors, replication vectors,
probe generation vectors, sequencing vectors, etc. In general, vectors of
interest may contain an origin of replication functional in at least one
organism, convenient restriction endonuclease sensitive sites, and
selectable markers for one or more host cell systems.

[0059]Another aspect of the subject invention is to provide for
PRCP-specific hybridization probes capable of hybridizing with naturally
occurring nucleotide sequences encoding PRCP. Such probes may also be
used for the detection of similar protease encoding sequences and should
preferably show at least 40% nucleotide identity to PRCP polynucleotides.
The hybridization probes of the subject invention may be derived from the
nucleotide sequence presented as SEQ ID NO: 1 or from genomic sequences
including promoter, enhancers or introns of the native gene.
Hybridization probes may be labelled by a variety of reporter molecules
using techniques well known in the art.

[0060]It will be recognized that many deletional or mutational analogs of
PRCP polynucleotides will be effective hybridization probes for PRCP
polynucleotides. Accordingly, the invention relates to nucleic acid
sequences that hybridize with such PRCP encoding nucleic acid sequences
under stringent conditions.

[0061]"Stringent conditions" refers to conditions that allow for the
hybridization of substantially related nucleic acid sequences. For
instance, such conditions will generally allow hybridization of sequence
with at least about 85% sequence identity, preferably with at least about
90% sequence identity, more preferably with at least about 95% sequence
identity. Hybridization conditions and probes can be adjusted in
well-characterized ways to achieve selective hybridization of
human-derived probes. Stringent conditions, within the meaning of the
invention are 68° C. in a buffer containing 0.2×SSC
(1× standard saline-citrate=150 mM NaCl, 15 mM Trinatriumcitrat)
[Sambrook et al., (1989)].

[0062]Nucleic acid molecules that will hybridize to PRCP polynucleotides
under stringent conditions can be identified functionally. Without
limitation, examples of the uses for hybridization probes include:
histochemical uses such as identifying tissues that express PRCP;
measuring mRNA levels, for instance to identify a sample's tissue type or
to identify cells that express abnormal levels of PRCP; and detecting
polymorphisms of PRCP.

[0063]PCR provides additional uses for oligonucleotides based upon the
nucleotide sequence which encodes PRCP. Such probes used in PCR may be of
recombinant origin, chemically synthesized, or a mixture of both.
Oligomers may comprise discrete nucleotide sequences employed under
optimized conditions for identification of PRCP in specific tissues or
diagnostic use. The same two oligomers, a nested set of oligomers, or
even a degenerate pool of oligomers may be employed under less stringent
conditions for identification of closely related DNAs or RNAs.

[0064]Rules for designing polymerase chain reaction (PCR) primers are now
established, as reviewed by PCR Protocols. Degenerate primers, i.e.,
preparations of primers that are heterogeneous at given sequence
locations, can be designed to amplify nucleic acid sequences that are
highly homologous to, but not identical with PRCP. Strategies are now
available that allow for only one of the primers to be required to
specifically hybridize with a known sequence. For example, appropriate
nucleic acid primers can be ligated to the nucleic acid sought to be
amplified to provide the hybridization partner for one of the primers. In
this way, only one of the primers need be based on the sequence of the
nucleic acid sought to be amplified.

[0065]PCR methods for amplifying nucleic acid will utilize at least two
primers. One of these primers will be capable of hybridizing to a first
strand of the nucleic acid to be amplified and of priming enzyme-driven
nucleic acid synthesis in a first direction. The other will be capable of
hybridizing the reciprocal sequence of the first strand (if the sequence
to be amplified is single stranded, this sequence will initially be
hypothetical, but will be synthesized in the first amplification cycle)
and of priming nucleic acid synthesis from that strand in the direction
opposite the first direction and towards the site of hybridization for
the first primer. Conditions for conducting such amplifications,
particularly under preferred stringent hybridization conditions, are well
known.

[0066]Other means of producing specific hybridization probes for PRCP
include the cloning of nucleic acid sequences encoding PRCP or PRCP
derivatives into vectors for the production of mRNA probes. Such vectors
are known in the art, are commercially available and may be used to
synthesize RNA probes in vitro by means of the addition of the
appropriate RNA polymerase as T7 or SP6 RNA polymerase and the
appropriate reporter molecules.

[0067]It is possible to produce a DNA sequence, or portions thereof,
entirely by synthetic chemistry. After synthesis, the nucleic acid
sequence can be inserted into any of the many available DNA vectors and
their respective host cells using techniques which are well known in the
art. Moreover, synthetic chemistry may be used to introduce mutations
into the nucleotide sequence. Alternately, a portion of sequence in which
a mutation is desired can be synthesized and recombined with longer
portion of an existing genomic or recombinant sequence.

[0068]PRCP polynucleotides may be used to produce a purified oligo- or
polypeptide using well known methods of recombinant DNA technology. The
oligopeptide may be expressed in a variety of host cells, either
prokaryotic or eukaryotic. Host cells may be from the same species from
which the nucleotide sequence was derived or from a different species.
Advantages of producing an oligonucleotide by recombinant DNA technology
include obtaining adequate amounts of the protein for purification and
the availability of simplified purification procedures.

Quantitative Determinations of Nucleic Acids

[0069]An important step in the molecular genetic analysis of human disease
is often the enumeration of the copy number of a nucleis acid or the
relative expression of a gene in particular tissues.

[0070]Several different approaches are currently available to make
quantitative determinations of nucleic acids. Chromosome-based
techniques, such as comparative genomic hybridization (CGH) and
fluorescent in situ hybridization (FISH) facilitate efforts to
cytogenetically localize genomic regions that are altered in tumor cells.
Regions of genomic alteration can be narrowed further using loss of
heterozygosity analysis (LOH), in which disease DNA is analyzed and
compared with normal DNA for the loss of a heterozygous polymorphic
marker. The first experiments used restriction fragment length
polymorphisms (RFLPs) [Johnson, (1989)], or hypervariable minisatellite
DNA [Barnes, 2000]. In recent years LOH has been performed primarily
using PCR amplification of microsatellite markers and electrophoresis of
the radio labelled [Jeffreys, (1985)] or fluorescently labelled PCR
products [Weber, (1990)] and compared between paired normal and disease
DNAs.

[0071]A number of other methods have also been developed to quantify
nucleic acids [Gergen, (1992)]. More recently, PCR and RT-PCR methods
have been developed which are capable of measuring the amount of a
nucleic acid in a sample. One approach, for example, measures PCR product
quantity in the log phase of the reaction before the formation of
reaction products plateaus [Thomas, (1980)].

[0072]A gene sequence contained in all samples at relatively constant
quantity is typically utilized for sample amplification efficiency
normalization. This approach, however, suffers from several drawbacks.
The method requires that each sample has equal input amounts of the
nucleic acid and that the amplification efficiency between samples is
identical until the time of analysis. Furthermore, it is difficult using
the conventional methods of PCR quantitation such as gel electrophoresis
or plate capture hybridization to determine that all samples are in fact
analyzed during the log phase of the reaction as required by the method.

[0073]Another method called quantitative competitive (QC)-PCR, as the name
implies, relies on the inclusion of an internal control competitor in
each reaction [Piatak, (1993), BioTechniques]. The efficiency of each
reaction is normalized to the internal competitor. A known amount of
internal competitor is typically added to each sample. The unknown target
PCR product is compared with the known competitor PCR product to obtain
relative quantitation. A difficulty with this general approach lies in
developing an internal control that amplifies with the same efficiency
than the target molecule.

5' Fluorogenic Nuclease Assays

[0074]Fluorogenic nuclease assays are a real time quantitation method that
uses a probe to monitor formation of amplification product. The basis for
this method of monitoring the formation of amplification product is to
measure continuously PCR product accumulation using a dual-labelled
fluorogenic oligonucleotide probe, an approach frequently referred to in
the literature simply as the "TaqMan method" [Piatak, (1993), Science;
Heid, (1996); Gibson, (1996); Holland. (1991)].

[0075]The probe used in such assays is typically a short (about 20-25
bases) oligonucleotide that is labeled with two different fluorescent
dyes. The 5' terminus of the probe is attached to a reporter dye and the
3' terminus is attached to a quenching dye, although the dyes could be
attached at other locations on the probe as well. The probe is designed
to have at least substantial sequence complementarity with the probe
binding site. Upstream and downstream PCR primers which bind to flanking
regions of the locus are added to the reaction mixture. When the probe is
intact, energy transfer between the two fluorophors occurs and the
quencher quenches emission from the reporter. During the extension phase
of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic
acid polymerase such as Taq polymerase, thereby releasing the reporter
from the oligonucleotide-quencher and resulting in an increase of
reporter emission intensity which can be measured by an appropriate
detector.

[0076]One detector which is specifically adapted for measuring
fluorescence emissions such as those created during a fluorogenic assay
is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in
Foster City, Calif. The ABI 7700 uses fiber optics connected with each
well in a 96- or 384 well PCR tube arrangement. The instrument includes a
laser for exciting the labels and is capable of measuring the
fluorescence spectra intensity from each tube with continuous monitoring
during PCR amplification. Each tube is re-examined every 8.5 seconds.

[0077]Computer software provided with the instrument is capable of
recording the fluorescence intensity of reporter and quencher over the
course of the amplification. The recorded values will then be used to
calculate the increase in normalized reporter emission intensity on a
continuous basis. The increase in emission intensity is plotted versus
time, i.e., the number of amplification cycles, to produce a continuous
measure of amplification. To quantify the locus in each amplification
reaction, the amplification plot is examined at a point during the log
phase of product accumu-lation. This is accomplished by assigning a
fluorescence threshold intensity above background and determining the
point at which each amplification plot crosses the threshold (defined as
the threshold cycle number or Ct). Differences in threshold cycle number
are used to quantify the relative amount of PCR target contained within
each tube. Assuming that each reaction functions at 100% PCR efficiency,
a difference of one Ct represents a two-fold difference in the amount of
starting template. The fluorescence value can be used in conjunction with
a standard curve to determine the amount of amplification product
present.

Non-Probe-Based Detection Methods

[0078]A variety of options are available for measuring the amplification
products as they are formed. One method utilizes labels, such as dyes,
which only bind to double stranded DNA. In this type of approach,
amplification product (which is double stranded) binds dye molecules in
solution to form a complex. With the appropriate dyes, it is possible to
distinguish between dye molecules free in solution and dye molecules
bound to amplification product. For example, certain dyes fluoresce only
when bound to amplification product. Examples of dyes which can be used
in methods of this general type include, but are not limited to, Syber
Green® and Pico Green from Molecular Probes, Inc. of Eugene, Oreg.,
ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst
33258, Toto-1, Yoyo-1, DAPI (4',6-diamidino-2-phenylindole
hydrochloride).

[0080]These detection methods involve some alteration to the structure or
conformation of a probe hybridized to the locus between the amplification
primer pair. In some instances, the alteration is caused by the
template-dependent extension catalyzed by a nucleic acid polymerase
during the amplification process. The alteration generates a detectable
signal which is an indirect measure of the amount of amplification
product formed.

[0081]For example, some methods involve the degradation or digestion of
the probe during the extension reaction. These methods are a consequence
of the 5'-3' nuclease activity associated with some nucleic acid
polymerases. Polymerases having this activity cleave mononucleotides or
small oligonucleotides from an oligonucleotide probe annealed to its
complementary sequence located within the locus.

[0082]The 3' end of the upstream primer provides the initial binding site
for the nucleic acid polymerase. As the polymerase catalyzes extension of
the upstream primer and encounters the bound probe, the nucleic acid
polymerase displaces a portion of the 5' end of the probe and through its
nuclease activity cleaves mononucleotides or oligonucleotides from the
probe.

[0083]The upstream primer and the probe can be designed such that they
anneal to the complementary strand in close proximity to one another. In
fact, the 3' end of the upstream primer and the 5' end of the probe may
abut one another. In this situation, extension of the upstream primer is
not necessary in order for the nucleic acid polymerase to begin cleaving
the probe. In the case in which intervening nucleotides separate the
upstream primer and the probe, extension of the primer is necessary
before the nucleic acid polymerase encounters the 5' end of the probe.
Once contact occurs and polymerization continues, the 5'-3' exonuclease
activity of the nucleic acid polymerase begins cleaving mononucleotides
or oligonucleotides from the 5' end of the probe. Digestion of the probe
continues until the remaining portion of the probe dissociates from the
complementary strand.

[0084]In solution, the two end sections can hybridize with each other to
form a hairpin loop. In this conformation, the reporter and quencher dye
are in sufficiently close proximity that fluorescence from the reporter
dye is effectively quenched by the quencher dye. Hybridized probe, in
contrast, results in a linearized conformation in which the extent of
quenching is decreased. Thus, by monitoring emission changes for the two
dyes, it is possible to indirectly monitor the formation of amplification
product.

Probes

[0085]The labeled probe is selected so that its sequence is substantially
complementary to a segment of the test locus or a reference locus. As
indicated above, the nucleic acid site to which the probe binds should be
located between the primer binding sites for the upstream and downstream
amplification primers.

Primers

[0086]The primers used in the amplification are selected so as to be
capable of hybridizing to sequences at flanking regions of the locus
being amplified. The primers are chosen to have at least substantial
complementarity with the different strands of the nucleic acid being
amplified. When a probe is utilized to detect the formation of
amplification products, the primers are selected in such that they flank
the probe, i.e. are located upstream and downstream of the probe.

[0087]The primer must have sufficient length so that it is capable of
priming the synthesis of extension products in the presence of an agent
for polymerization. The length and composition of the primer depends on
many parameters, including, for example, the temperature at which the
annealing reaction is conducted, proximity of the probe binding site to
that of the primer, relative concentrations of the primer and probe and
the particular nucleic acid composition of the probe. Typically the
primer includes 15-30 nucleotides. However, the length of the primer may
be more or less depending on the complexity of the primer binding site
and the factors listed above.

Labels for Probes and Primers

[0088]The labels used for labeling the probes or primers of the current
invention and which can provide the signal corresponding to the quantity
of amplification product can take a variety of forms. As indicated above
with regard to the 5' fluorogenic nuclease method, a fluorescent signal
is one signal which can be measured. However, measurements may also be
made, for example, by monitoring radioactivity, colorimetry, absorption,
magnetic parameters, or enzymatic activity. Thus, labels which can be
employed include, but are not limited to, fluorophors, chromophores,
radioactive isotopes, electron dense reagents, enzymes, and ligands
having specific binding partners (e.g., biotin-avidin).

[0089]Monitoring changes in fluorescence is a particularly useful way to
monitor the accumulation of amplification products. A number of labels
useful for attachment to probes or primers are commercially available
including fluorescein and various fluorescein derivatives such as FAM,
HEX, TET and JOE (all which are available from Applied Biosystems, Foster
City, Calif.); lucifer yellow, and coumarin derivatives.

[0090]Labels may be attached to the probe or primer using a variety of
techniques and can be attached at the 5' end, and/or the 3' end and/or at
an internal nucleotide. The label can also be attached to spacer arms of
various sizes which are attached to the probe or primer. These spacer
arms are useful for obtaining a desired distance between multiple labels
attached to the probe or primer.

[0091]In some instances, a single label may be utilized; whereas, in other
instances, such as with the 5' fluorogenic nuclease assays for example,
two or more labels are attached to the probe. In cases wherein the probe
includes multiple labels, it is generally advisable to maintain spacing
between the labels which is sufficient to permit separation of the labels
during digestion of the probe through the 5'-3' nuclease activity of the
nucleic acid polymerase.

Patients Exhibiting Symptoms of Disease

[0092]A number of diseases are associated with changes in the copy number
of a certain gene. For patients having symptoms of a disease, the
real-time PCR method can be used to determine if the patient has copy
number alterations which are known to be linked with diseases that are
associated with the symptoms the patient has.

PRCP Expression

PRCP Fusion Proteins

[0093]Fusion proteins are useful for generating antibodies against PRCP
polypeptides and for use in various assay systems. For example, fusion
proteins can be used to identify proteins which interact with portions of
PRCP polypeptides. Protein affinity chromatography or library-based
assays for protein-protein interactions, such as the yeast two-hybrid or
phage display systems, can be used for this purpose. Such methods are
well known in the art and also can be used as drug screens.

[0094]A PRCP fusion protein comprises two polypeptide segments fused
together by means of a peptide bond. The first polypeptide segment can
comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, 275,
300, 325 or 350 contiguous amino acids of SEQ ID NO: 2 or of a
biologically active variant, such as those described above. The first
polypeptide segment also can comprise full-length PRCP.

[0096]A naturally occurring PRCP polynucleotide can be isolated free of
other cellular components such as membrane components, proteins, and
lipids. Polynucleotides can be made by a cell and isolated using standard
nucleic acid purification techniques, or synthesized using an
amplification technique, such as the polymerase chain reaction (PCR), or
by using an automatic synthesizer. Methods for isolating polynucleotides
are routine and are known in the art. Any such technique for obtaining a
polynucleotide can be used to obtain isolated PRCP polynucleotides. For
example, restriction enzymes and probes can be used to isolate
polynucleotide fragments which comprise PRCP nucleotide sequences.
Isolated polynucleotides are in preparations which are free or at least
70, 80, or 90% free of other molecules.

[0097]PRCP cDNA molecules can be made with standard molecular biology
techniques, using PRCP mRNA as a template. PRCP cDNA molecules can
thereafter be replicated using molecular biology techniques known in the
art. An amplification technique, such as PCR, can be used to obtain
additional copies of polynucleotides of the invention, using either human
genomic DNA or cDNA as a template.

[0098]Alternatively, synthetic chemistry techniques can be used to
synthesizes PRCP polynucleotides. The degeneracy of the genetic code
allows alternate nucleotide sequences to be synthesized which will encode
PRCP having, for example, an amino acid sequence shown in SEQ ID NO: 2 or
a biologically active variant thereof.

Extending Polynucleotides

[0099]Various PCR-based methods can be used to extend nucleic acid
sequences encoding human PRCP, for example to detect upstream sequences
of PRCP gene such as promoters and regulatory elements. For example,
restriction-site PCR uses universal primers to retrieve unknown sequence
adjacent to a known locus. Genomic DNA is first amplified in the presence
of a primer to a linker sequence and a primer specific to the known
region. The amplified sequences are then subjected to a second round of
PCR with the same linker primer and another specific primer internal to
the first one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse transcriptase.

[0100]Inverse PCR also can be used to amplify or extend sequences using
divergent primers based on a known region. Primers can be designed using
commercially available software, such as OLIGO 4.06 Primer Analysis
software (National Biosciences Inc., Plymouth, Minn.), to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to anneal
to the target sequence at temperatures about 68-72° C. The method
uses several restriction enzymes to generate a suitable fragment in the
known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template.

[0101]Another method which can be used is capture PCR, which involves PCR
amplification of DNA fragments adjacent to a known sequence in human and
yeast artificial chromosome DNA. In this method, multiple restriction
enzyme digestions and ligations also can be used to place an engineered
double-stranded sequence into an unknown fragment of the DNA molecule
before performing PCR.

[0102]When screening for full-length cDNAs, it is preferable to use
libraries that have been size-selected to include larger cDNAs.
Randomly-primed libraries are preferable, in that they will contain more
sequences which contain the 5' regions of genes. Use of a randomly primed
library may be especially preferable for situations in which an oligo
d(T) library does not yield a full-length cDNA. Genomic libraries can be
useful for extension of sequence into 5' non-transcribed regulatory
regions.

[0103]Commercially available capillary electrophoresis systems can be used
to analyze the size or confirm the nucleotide sequence of PCR or
sequencing products. For example, capillary sequencing can employ
flowable polymers for electrophoretic separation, four different
fluorescent dyes (one for each nucleotide) which are laser activated, and
detection of the emitted wavelengths by a charge coupled device camera.
Output/light intensity can be converted to electrical signal using
appropriate equipment and software (e.g., GENOTYPER and Sequence
NAVIGATOR, Perkin Elmer), and the entire process from loading of samples
to computer analysis and electronic data display can be computer
controlled. Capillary electrophoresis is especially preferable for the
sequencing of small pieces of DNA which might be present in limited
amounts in a particular sample.

Obtaining Polypeptides

[0104]PRCP can be obtained, for example, by purification from human cells,
by expression of PRCP polynucleotides, or by direct chemical synthesis.

Protein Purification

[0105]PRCP can be purified from any human cell which expresses the enzyme,
including those which have been transfected with expression constructs
which express PRCP. A purified PRCP is separated from other compounds
which normally associate with PRCP in the cell, such as certain proteins,
carbohydrates, or lipids, using methods well-known in the art. Such
methods include, but are not limited to, size exclusion chromatography,
ammonium sulfate fractionation, ion exchange chromatography, affinity
chromatography, and preparative gel electrophoresis.

Expression of PRCP Polynucleotides

[0106]To express PRCP, PRCP polynucleotides can be inserted into an
expression vector which contains the necessary elements for the
transcription and translation of the inserted coding sequence. Methods
which are well known to those skilled in the art can be used to construct
expression vectors containing sequences encoding PRCP and appropriate
transcriptional and translational control elements. These methods include
in vitro recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination.

[0108]The control elements or regulatory sequences are those
non-translated regions of the vector--enhancers, promoters, 5' and 3'
untranslated regions--which interact with host cellular proteins to carry
out transcription and translation. Such elements can vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation elements,
including constitutive and inducible promoters, can be used. For example,
when cloning in bacterial systems, inducible promoters such as the hybrid
lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or
pSPORT1 plasmid (Life Technologies) and the like can be used. The
baculovirus polyhedrin promoter can be used in insect cells. Promoters or
enhancers derived from the genomes of plant cells (e.g., heat shock,
RUBISCO, and storage protein genes) or from plant viruses (e.g., viral
promoters or leader sequences) can be cloned into the vector. In
mammalian cell systems, promoters from mammalian genes or from mammalian
viruses are preferable. If it is necessary to generate a cell line that
contains multiple copies of a nucleotide sequence encoding PRCP, vectors
based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

[0109]In bacterial systems, a number of expression vectors can be
selected. For example, when a large quantity of PRCP is needed for the
induction of antibodies, vectors which direct high level expression of
fusion proteins that are readily purified can be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning and
expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT
vector, a sequence encoding PRCP can be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7 residues
of β-galactosidase so that a hybrid protein is produced. pIN vectors
or pGEX vectors (Promega, Madison, Wis.) also can be used to express
foreign polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-agarose beads
followed by elution in the presence of free glutathione. Proteins made in
such systems can be designed to include heparin, thrombin, or factor Xa
protease cleavage sites so that the cloned polypeptide of interest can be
released from the GST moiety at will.

Plant and Insect Expression Systems

[0110]If plant expression vectors are used, the expression of sequences
encoding PRCP can be driven by any of a number of promoters. For example,
viral promoters such as the 35S and 19S promoters of CaMV can be used
alone or in combination with the omega leader sequence from TMV.
Alternatively, plant promoters such as the small subunit of RUBISCO or
heat shock promoters can be used. These constructs can be introduced into
plant cells by direct DNA transformation or by pathogen-mediated
transfection.

[0111]An insect system also can be used to express PRCP. For example, in
one such system Autographa califormica nuclear polyhedrosis virus (AcNPV)
is used as a vector to express foreign genes in Spodoptera frugiperda
cells or in Trichoplusia larvae. Sequences encoding PRCP can be cloned
into a non-essential region of the virus, such as the polyhedrin gene,
and placed under control of the polyhedrin promoter. Successful insertion
of PRCP will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein. The recombinant viruses can then be used to
infect S. frugiperda cells or Trichoplusia larvae in which PRCP can be
expressed.

Mammalian Expression Systems

[0112]A number of viral-based expression systems can be used to express
PRCP in mammalian host cells. For example, if an adenovirus is used as an
expression vector, sequences encoding PRCP can be ligated into an
adenovirus transcription/translation complex comprising the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or E3
region of the viral genome can be used to obtain a viable virus which is
capable of expressing PRCP in infected host cells [Engelhard, 1994)]. If
desired, transcription enhancers, such as the Rous sarcoma virus (RSV)
enhancer, can be used to increase expression in mammalian host cells.

[0113]Human artificial chromosomes (HACs) also can be used to deliver
larger fragments of DNA than can be contained and expressed in a plasmid.
HACs of 6M to 10M are constructed and delivered to cells via conventional
delivery methods (e.g., liposomes, polycationic amino polymers, or
vesicles). Specific initiation signals also can be used to achieve more
efficient translation of sequences encoding PRCP. Such signals include
the ATG initiation codon and adjacent sequences. In cases where sequences
encoding PRCP, its initiation codon, and upstream sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals may be needed. However, in cases where only
coding sequence, or a fragment thereof, is inserted, exogenous
translational control signals (including the ATG initiation codon) should
be provided. The initiation codon should be in the correct reading frame
to ensure translation of the entire insert. Exogenous translational
elements and initiation codons can be of various origins, both natural
and synthetic.

Host Cells

[0114]A host cell strain can be chosen for its ability to modulate the
expression of the inserted sequences or to process the expressed PRCP in
the desired fashion. Such modifications of the polypeptide include, but
are not limited to, acetylation, carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" form of the polypeptide also can be used to
facilitate correct insertion, folding and/or function. Different host
cells which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK,
HEK293, and W138), are available from the American Type Culture
Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209)
and can be chosen to ensure the correct modification and processing of
the foreign protein.

[0115]Stable expression is preferred for long-term, high-yield production
of recombinant proteins. For example, cell lines which stably express
PRCP can be transformed using expression vectors which can contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector. Following the
introduction of the vector, cells can be allowed to grow for 1-2 days in
an enriched medium before they are switched to a selective medium. The
purpose of the selectable marker is to confer resistance to selection,
and its presence allows growth and recovery of cells which successfully
express the introduced PRCP sequences. Resistant clones of stably
transformed cells can be proliferated using tissue culture techniques
appropriate to the cell type. Any number of selection systems can be used
to recover transformed cell lines. These include, but are not limited to,
the herpes simplex virus thymidine kinase [Logan, (1984)] and adenine
phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in
tk.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate [Lowy,
(1980)], npt confers resistance to the aminoglycosides, neomycin and
G-418 [Wigler, (1980)], and als and pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively
[Colbere-Garapin, 1981]. Additional selectable genes have been described.
For example, trpB allows cells to utilize indole in place of tryptophan,
or hisD, which allows cells to utilize histinol in place of histidine.
Visible markers such as anthocyanins, β-glucuronidase and its
substrate GUS, and luciferase and its substrate luciferin, can be used to
identify transformants and to quantify the amount of transient or stable
protein expression attributable to a specific vector system

Detecting Polypeptide Expression

[0116]Although the presence of marker gene expression suggests that a PRCP
polynucleotide is also present, its presence and expression may need to
be confirmed. For example, if a sequence encoding PRCP is inserted within
a marker gene sequence, transformed cells containing sequences which
encode PRCP can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence
encoding PRCP under the control of a single promoter. Expression of the
marker gene in response to induction or selection usually indicates
expression of PRCP polynucleotide.

[0117]Alternatively, host cells which contain a PRCP polynucleotide and
which express PRCP can be identified by a variety of procedures known to
those of skill in the art. These procedures include, but are not limited
to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip-based technologies
for the detection and/or quantification of nucleic acid or protein. For
example, the presence of a poly-nucleotide sequence encoding PRCP can be
detected by DNA-DNA or DNA-RNA hybridization or amplification using
probes or fragments or fragments of polynucleotides encoding PRCP.
Nucleic acid amplification-based assays involve the use of
oligonucleotides selected from sequen-ces encoding PRCP to detect
transformants which contain a PRCP polynucleotide.

[0118]A variety of protocols for detecting and measuring the expression of
PRCP, using either polyclonal or monoclonal antibodies specific for the
polypeptide, are known in the art. Examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
using monoclonal antibodies reactive to two non-interfering epitopes on
PRCP can be used, or a competitive binding assay can be employed.

[0119]A wide variety of labels and conjugation techniques are known by
those skilled in the art and can be used in various nucleic acid and
amino acid assays. Means for producing labeled hybridization or PCR
probes for detecting sequences related to polynucleotides encoding PRCP
include oligolabeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide. Alternatively, sequences
encoding PRCP can be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially available, and
can be used to synthesize RNA probes in vitro by addition of labeled
nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6.
These procedures can be conducted using a variety of commercially
available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
Suitable reporter molecules or labels which can be used for ease of
detection include radionuclides, enzymes, and fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

[0120]Host cells transformed with PRCP polynucleotides can be cultured
under conditions suitable for the expression and recovery of the protein
from cell culture. The polypeptide produced by a transformed cell can be
secreted or contained intracellularly depending on the sequence and/or
the vector used. As will be understood by those of skill in the art,
expression vectors containing PRCP polynucleotides can be designed to
contain signal sequences which direct secretion of soluble PRCP through a
prokaryotic or eukaryotic cell membrane or which direct the membrane
insertion of membrane-bound PRCP.

[0121]As discussed above, other constructions can be used to join a
sequence encoding PRCP to a nucleotide sequence encoding a polypeptide
domain which will facilitate purification of soluble proteins. Such
purification facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized in
the FLAGS extension/affinity purification system (Immunex Corp., Seattle,
Wash.). Inclusion of cleavable linker sequences such as those specific
for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the
purification domain and PRCP also can be used to facilitate purification.
One such expression vector provides for expression of a fusion protein
containing PRCP and 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification by IMAC (immobilized metal ion affinity chromatography)
Maddox, (1983)], while the enterokinase cleavage site provides a means
for purifying PRCP from the fusion protein [Porath, (1992)].

Chemical Synthesis

[0122]Sequences encoding PRCP can be synthesized, in whole or in part,
using chemical methods well known in the art. Alternatively, PRCP itself
can be produced using chemical methods to synthesize its amino acid
sequence, such as by direct peptide synthesis using solid-phase
techniques. Protein synthesis can either be performed using manual
techniques or by automation. Automated synthesis can be achieved, for
example, using Applied Biosystems 431A Peptide Synthesizer (Perkin
Elmer). Optionally, fragments of PRCP can be separately synthesized and
combined using chemical methods to produce a full-length molecule.

[0123]The newly synthesized peptide can be substantially purified by
preparative high performance liquid chromatography. The composition of a
synthetic PRCP can be confirmed by amino acid analysis or sequencing.
Additionally, any portion of the amino acid sequence of PRCP can be
altered during direct synthesis and/or combined using chemical methods
with sequences from other proteins to produce a variant polypeptide or a
fusion protein.

Production of Altered Polypeptides

[0124]As will be understood by those of skill in the art, it may be
advantageous to produce PRCP polynucleotides possessing non-naturally
occurring codons. For example, codons preferred by a particular
prokaryotic or eukaryotic host can be selected to increase the rate of
protein expression or to produce an RNA transcript having desirable
properties, such as a half-life which is longer than that of a transcript
generated from the naturally occurring sequence.

[0125]The nucleotide sequences referred to herein can be engineered using
methods generally known in the art to alter PRCP polynucleotides for a
variety of reasons, including but not limited to, alterations which
modify the cloning, processing, and/or expression of the polypeptide or
mRNA product. DNA shuffling by random fragmentation and PCR reassembly of
gene fragments and synthetic oligonucleotides can be used to engineer the
nucleotide sequences. For example, site-directed mutagenesis can be used
to insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and so
forth.

PRCP Analogs

[0126]One general class of PRCP analogs are variants having an amino acid
sequence that is a mutation of the amino acid sequence disclosed herein.
Another general class of PRCP analogs is provided by anti-idiotype
antibodies, and fragments thereof, as described below. Moreover,
recombinant antibodies comprising anti-idiotype variable domains can be
used as analogs (see, for example, [Monfardini et al., (1996)]). Since
the variable domains of anti-idiotype PRCP antibodies mimic PRCP, these
domains can provide PRCP enzymatic activity. Methods of producing
anti-idiotypic catalytic antibodies are known to those of skill in the
art [Joron et al., (1992), Friboulet et al. (1994), Avalle et al.,
(1998)].

[0127]Another approach to identifying PRCP analogs is provided by the use
of combinatorial libraries. Methods for constructing and screening phage
display and other combinatorial libraries are provided, for example, by
[Kay et al., Phage Display of Peptides and Proteins (Academic Press
1996), U.S. Pat. No. 5,783,384, U.S. Pat. No. 5,747,334, and U.S. Pat.
No. 5,723,323.

[0128]One illustrative in vitro use of PRCP and its analogs is the
production of labeled peptides from a labeled protein substrate.
Proteases can also be used in detergents and cleaning solutions. For
example, serine proteases are used in solutions to clean and to disinfect
contact lenses (see, for example, [U.S. Pat. No. 5,985,629]). Another use
for a serine protease is in the formulation of vaccines (see, for
example, [U.S. Pat. No. 5,885,814]). Those of skill in the art can devise
other uses for molecules having PRCP activity.

Antibodies

[0129]Any type of antibody known in the art can be generated to bind
specifically to an epitope of PRCP.

[0130]"Antibody" as used herein includes intact immunoglobulin molecules,
as well as fragments thereof, such as Fab, F(ab')2, and Fv, which
are capable of binding an epitope of PRCP. Typically, at least 6, 8, 10,
or 12 contiguous amino acids are required to form an epitope. However,
epitopes which involve non-contiguous amino acids may require more, e.g.,
at least 15, 25, or 50 amino acid. An antibody which specifically binds
to an epitope of PRCP can be used therapeutically, as well as in
immunochemical assays, such as Western blots, ELISAs, radioimmunoassays,
immunohistochemical assays, immunoprecipitations, or other immunochemical
assays known in the art. Various immunoassays can be used to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays are well known in the
art. Such immunoassays typically involve the measurement of complex
formation between an immunogen and an antibody which specifically binds
to the PRCP immunogen.

[0131]Typically, an antibody which specifically binds to PRCP provides a
detection signal at least 5-, 10-, or 20-fold higher than a detection
signal provided with other proteins when used in an immunochemical assay.
Preferably, antibodies which specifically bind to PRCP do not detect
other proteins in immunochemical assays and can immunoprecipitate PRCP
from solution.

[0132]PRCP can be used to immunize a mammal, such as a mouse, rat, rabbit,
guinea pig, monkey, or human, to produce polyclonal antibodies. If
desired, PRCP can be conjugated to a carrier protein, such as bovine
serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on
the host species, various adjuvants can be used to increase the
immunological response. Such adjuvants include, but are not limited to,
Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface
active substances (e.g., lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially useful.

[0133]Monoclonal antibodies which specifically bind to PRCP can be
prepared using any technique which provides for the production of
antibody molecules by continuous cell lines in culture. These techniques
include, but are not limited to, the hybridoma technique, the human
B-cell hybridoma technique, and the EBV-hybridoma technique [Roberge,
(1995)].

[0134]In addition, techniques developed for the production of "chimeric
antibodies", the splicing of mouse antibody genes to human antibody genes
to obtain a molecule with appropriate antigen specificity and biological
activity, can be used. Monoclonal and other antibodies also can be
"humanized" to prevent a patient from mounting an immune response against
the antibody when it is used therapeutically. Such antibodies may be
sufficiently similar in sequence to human antibodies to be used directly
in therapy or may require alteration of a few key residues. Sequence
differences between rodent antibodies and human sequences can be
minimized by replacing residues which differ from those in the human
sequences by site directed mutagenesis of individual residues or by
grating of entire complementarity determining regions. Antibodies which
specifically bind to PRCP can contain antigen binding sites which are
either partially or fully humanized, as disclosed in U.S. Pat. No.
5,565,332.

[0135]Alternatively, techniques described for the production of single
chain antibodies can be adapted using methods known in the art to produce
single chain antibodies which specifically bind to PRCP. Antibodies with
related specificity, but of distinct idiotypic composition, can be
generated by chain shuffling from random combinatorial immunoglobin
libraries. Single-chain antibodies also can be constructed using a DNA
amplification method, such as PCR, using hybridoma cDNA as a template.
Single-chain antibodies can be mono- or bispecific, and can be bivalent
or tetravalent. Construction of tetravalent, bispecific single-chain
antibodies is taught. A nucleotide sequence encoding a single-chain
antibody can be constructed using manual or automated nucleo-tide
synthesis, cloned into an expression construct using standard recombinant
DNA methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology.

[0136]Antibodies which specifically bind to PRCP also can be produced by
inducing in vivo production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding reagents.
Other types of antibodies can be constructed and used therapeutically in
methods of the invention. For example, chimeric antibodies can be
constructed as disclosed in WO 93/03151. Binding proteins which are
derived from immunoglobulins and which are multivalent and multispecific,
such as the "diabodies" described in WO 94/13804, also can be prepared.

[0137]Antibodies according to the invention can be purified by methods
well known in the art. For example, antibodies can be affinity purified
by passage over a column to which PRCP is bound. The bound antibodies can
then be eluted from the column using a buffer with a high salt
concentration.

Antisense Oligonucleotides

[0138]Antisense oligonucleotides are nucleotide sequences which are
complementary to a specific DNA or RNA sequence. Once introduced into a
cell, the complementary nucleotides combine with natural sequences
produced by the cell to form complexes and block either transcription or
translation. Preferably, an antisense oligonucleotide is at least 11
nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,
45, or 50 or more nucleotides long. Longer sequences also can be used.
Antisense oligonucleotide molecules can be provided in a DNA construct
and introduced into a cell as described above to decrease the level of
PRCP gene products in the cell.

[0139]Antisense oligonucleotides can be deoxyribonucleotides,
ribonucleotides, or a combination of both. Oligonucleotides can be
synthesized manually or by an automated synthesizer, by covalently
linking the 5' end of one nucleotide with the 3' end of another
nucleotide with non-phosphodiester internucleotide linkages such
alkylphosphonates, phosphorothioates, phosphorodithioates,
alkyl-phosphonothioates, alkylphosphonates, phosphoramidates, phosphate
esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and
phosphate triesters.

[0140]Modifications of PRCP gene expression can be obtained by designing
antisense oligonucleotides which will form duplexes to the control, 5',
or regulatory regions of the PRCP gene. Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10 from
the start site, are preferred. Similarly, inhibition can be achieved
using "triple helix" base-pairing methodology. Triple helix pairing is
useful because it causes inhibition of the ability of the double helix to
open sufficiently for the binding of polymerases, transcription factors,
or chaperons. Therapeutic advances using triplex DNA have been described
in the literature

[0141][Nicholls, (1993)]. An antisense oligonucleotide also can be
designed to block translation of mRNA by preventing the transcript from
binding to ribosomes.

[0142]Precise complementarity is not required for successful complex
formation between an antisense oligonucleotide and the complementary
sequence of a PRCP polynucleotide. Antisense oligonucleotides which
comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous
nucleotides which are precisely complementary to a PRCP polynucleotide,
each separated by a stretch of contiguous nucleotides which are not
complementary to adjacent PRCP nucleotides, can provide sufficient
targeting specificity for PRCP mRNA. Preferably, each stretch of
comple-mentary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or
more nucleotides in length. Non-complementary intervening sequences are
preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art
can easily use the calculated melting point of an antisense-sense pair to
determine the degree of mismatching which will be tolerated between a
particular antisense oligonucleotide and a particular PRCP polynucleotide
sequence. Antisense oligonucleotides can be modified without affecting
their ability to hybridize to a PRCP polynucleotide. These modifications
can be internal or at one or both ends of the antisense molecule. For
example, internucleoside phosphate linkages can be modified by adding
cholesteryl or diamine moieties with varying numbers of carbon residues
between the amino groups and terminal ribose. Modified bases and/or
sugars, such as arabinose instead of ribose, or a 3',5'-substituted
oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group
are substituted, also can be employed in a modified antisense
oligo-nucleotide. These modified oligonucleotides can be prepared by
methods well known in the art.

Ribozymes

[0143]Ribozymes are RNA molecules with catalytic activity [Uhlmann,
(1987)]. Ribozymes can be used to inhibit gene function by cleaving an
RNA sequence, as is known in the art. The mechanism of ribozyme action
involves sequence-specific hybridization of the ribozyme molecule to
complementary target RNA, followed by endonucleolytic cleavage. Examples
include engineered hammerhead motif ribozyme molecules that can
specifically and efficiently catalyze endonucleo-lytic cleavage of
specific nucleotide sequences. The coding sequence of a PRCP
polynucleotide can be used to generate ribozymes which will specifically
bind to mRNA transcribed from a PRCP polynucleotide. Methods of designing
and constructing ribozymes which can cleave other RNA molecules in trans
in a highly sequence specific manner have been developed and described in
the art. For example, the cleavage activity of ribozymes can be targeted
to specific RNAs by engineering a discrete "hybridization" region into
the ribozyme. The hybridization region contains a sequence complementary
to the target RNA and thus specifically hybridizes with the target RNA.

[0144]Specific ribozyme cleavage sites within a PRCP RNA target can be
identified by scanning the target molecule for ribozyme cleavage sites
which include the following sequences: GUA, GUU, and GUC. Once
identified, short RNA sequences of between 15 and 20 ribonucleotides
corresponding to the region of the target RNA containing the cleavage
site can be evaluated for secondary structural features which may render
the target inoperable. Suitability of candidate PRCP RNA targets also can
be evaluated by testing accessibility to hybridization with complementary
oligonucleotides using ribonuclease protection assays. The nucleotide
sequences shown in SEQ ID NO: 1 and its complement provide sources of
suitable hybridization region sequences. Longer complementary sequences
can be used to increase the affinity of the hybridization sequence for
the target. The hybridizing and cleavage regions of the ribozyme can be
integrally related such that upon hybridizing to the target RNA through
the complementary regions, the catalytic region of the ribozyme can
cleave the target.

[0145]Ribozymes can be introduced into cells as part of a DNA construct.
Mechanical methods, such as microinjection, liposome-mediated
transfection, electroporation, or calcium phosphate precipitation, can be
used to introduce a ribozyme-containing DNA construct into cells in which
it is desired to decrease PRCP expression. Alternatively, if it is
desired that the cells stably retain the DNA construct, the construct can
be supplied on a plasmid and maintained as a separate element or
integrated into the genome of the cells, as is known in the art. A
ribozyme-encoding DNA construct can include transcriptional regulatory
elements, such as a promoter element, an enhancer or UAS element, and a
transcriptional terminator signal, for controlling transcription of
ribozymes in the cells (U.S. Pat. No. 5,641,673). Ribozymes also can be
engineered to provide an additional level of regulation, so that
destruction of mRNA occurs only when both a ribozyme and a target gene
are induced in the cells.

Screening/Screening Assays

Regulators

[0146]Regulators as used herein, refer to compounds that affect the
activity of PRCP in vivo and/or in vitro. Regulators can be agonists and
antagonists of PRCP polypeptide and can be compounds that exert their
effect on the PRCP activity via the enzymatic activity, expression,
post-translational modifications or by other means. Agonists of PRCP are
molecules which, when bound to PRCP, increase or prolong the activity of
PRCP. Agonists of PRCP include proteins, nucleic acids, carbohydrates,
small molecules, or any other molecule which activate PRCP. Antagonists
of PRCP are molecules which, when bound to PRCP, decrease the amount or
the duration of the activity of PRCP. Antagonists include proteins,
nucleic acids, carbohydrates, antibodies, small molecules, or any other
molecule which decrease the activity of PRCP.

[0147]The term "modulate", as it appears herein, refers to a change in the
activity of PRCP polypeptide. For example, modulation may cause an
increase or a decrease in enzymatic activity, binding characteristics, or
any other biological, functional, or immunological properties of PRCP.

[0148]As used herein, the terms "specific binding" or "specifically
binding" refer to that interaction between a protein or peptide and an
agonist, an antibody, or an antagonist. The interaction is dependent upon
the presence of a particular structure of the protein recognized by the
binding molecule (i.e., the antigenic determinant or epitope). For
example, if an antibody is specific for epitope "A" the presence of a
polypeptide containing the epitope A, or the presence of free unlabeled
A, in a reaction containing free labeled A and the antibody will reduce
the amount of labeled A that binds to the antibody.

[0149]The invention provides methods (also referred to herein as
"screening assays") for identifying compounds which can be used for the
treatment of diseases related to PRCP. The methods entail the
identification of candidate or test compounds or agents (e.g., peptides,
peptidomimetics, small molecules or other molecules) which bind to PRCP
and/or have a stimulatory or inhibitory effect on the biological activity
of PRCP or its expression and then determining which of these compounds
have an effect on symptoms or diseases related to PRCP in an in vivo
assay.

[0150]Candidate or test compounds or agents which bind to PRCP and/or have
a stimulatory or inhibitory effect on the activity or the expression of
PRCP are identified either in assays that employ cells which express PRCP
(cell-based assays) or in assays with isolated PRCP (cell-free assays).
The various assays can employ a variety of variants of PRCP (e.g.,
full-length PRCP, a biologically active fragment of PRCP, or a fusion
protein which includes all or a portion of PRCP). Moreover, PRCP can be
derived from any suitable mammalian species (e.g., human PRCP, rat PRCP
or murine PRCP). The assay can be a binding assay entailing direct or
indirect measurement of the binding of a test compound or a known PRCP
ligand to PRCP. The assay can also be an activity assay entailing direct
or indirect measurement of the activity of PRCP. The assay can also be an
expression assay entailing direct or indirect measurement of the
expression of PRCP mRNA or PRCP protein. The various screening assays are
combined with an in vivo assay entailing measuring the effect of the test
compound on the symptoms of diseases related to PRCP.

[0151]The present invention includes biochemical, cell free assays that
allow the identification of inhibitors and agonists of proteases suitable
as lead structures for pharmacological drug development. Such assays
involve contacting a form of PRCP (e.g., full-length PRCP, a biologically
active fragment of PRCP, or a fusion protein comprising all or a portion
of PRCP) with a test compound and determining the ability of the test
compound to act as an antagonist (preferably) or an agonist of the
enzymatic activity of PRCP.

[0152]The activity of PRCP molecules of the present invention can be
measured using a variety of assays that measure PRCP activity. For
example, PRCP enzyme activity can be assessed by a standard in vitro
serine/metallo/ . . . protease assay (see, for example, [U.S. Pat. No.
5,057,414]). Those of skill in the art are aware of a variety of
substrates suitable for in vitro assays, such as SucAla-Ala-Pro-Phe-pNA,
fluorescein mono-p-guanidinobenzoate hydrochloride,
benzyloxycarbonyl-L-Arginyl-5-benzyl-ester, Nalpha-Benzoyl-L-arginine
ethyl ester hydrochloride, and the like. In addition, protease assay kits
available from commercial sources, such as Calbiochem (San Diego,
Calif.). For general references, see Barrett (Ed.), Methods in
Enzymology, Proteolytic Enzymes: Serine and Cysteine Peptidases (Academic
Press Inc. 1994), and Barrett et al., (Eds.), Handbook of Proteolytic
Enzymes (Academic Press Inc. 1998).

[0153]Solution in vitro assays can be used to identify a PRCP substrate or
inhibitor. Solid phase systems can also be used to identify a substrate
or inhibitor of a PRCP polypeptide. For example, a PRCP polypeptide or
PRCP fusion protein can be immobilized onto the surface of a receptor
chip of a commercially available biosensor instrument (BIACORE, Biacore
AB; Uppsala, Sweden). The use of this instrument is disclosed, for
example, by [Karlsson, (1991), and Cunningham and Wells, (1993)].

[0154]In brief, a PRCP polypeptide or fusion protein is covalently
attached, using amine or sulfhydryl chemistry, to dextran fibers that are
attached to gold film within a flow cell. A test sample is then passed
through the cell. If a PRCP substrate or inhibitor is present in the
sample, it will bind to the immobilized polypeptide or fusion protein,
causing a change in the refractive index of the medium, which is detected
as a change in surface plasmon resonance of the gold film. This system
allows the determination on- and off-rates, from which binding affinity
can be calculated, and assessment of the stoichiometry of binding, as
well as the kinetic effects of PRCP mutation. This system can also be
used to examine antibody-antigen interactions, and the interactions of
other comple-ment/anti-complement pairs.

[0155]In one embodiment, the invention provides assays for screening
candidate or test compounds which bind to or modulate the activity of
PRCP. Such assays can employ full-length PRCP, a biologically active
fragment of PRCP, or a fusion protein which includes all or a portion of
PRCP. As described in greater detail below, the test compound can be
obtained by any suitable means, e.g., from conventional compound
libraries.

[0156]Determining the ability of the test compound to modulate the
activity of PRCP can be accomplished, for example, by determining the
ability of PRCP to bind to or interact with a target molecule. The target
molecule can be a molecule with which PRCP binds or interacts with in
nature. The target molecule can be a component of a signal transduction
pathway which facilitates transduction of an extracellular signal. The
target PRCP molecule can be, for example, a second intracellular protein
which has catalytic activity or a protein which facilitates the
association of downstream signaling molecules with PRCP.

[0157]Determining the ability of PRCP to bind to or interact with a target
molecule can be accomplished by one of the methods described above for
determining direct binding. In one embodiment, determining the ability of
a polypeptide of the invention to bind to or interact with a target
molecule can be accomplished by determining the activity of the target
molecule. For example, the activity of the target molecule can be
determined by detecting induction of a cellular second messenger of the
target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.),
detecting catalytic/enzy-uratic activity of the target on an appropriate
substrate, detecting the induction of a reporter gene (e.g., a regulatory
element that is responsive to a polypeptide of the invention operably
linked to a nucleic acid encoding a detectable marker, e.g., luciferase),
or detecting a cellular response.

[0158]In various embodiments of the above assay methods of the present
invention, it may be desirable to immobilize PRCP (or a PRCP target
molecule) to facilitate separation of complexed from uncomplexed forms of
one or both of the proteins, as well as to accommodate automation of the
assay. Binding of a test compound to PRCP, or interaction of PRCP with a
target molecule in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtitre plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the proteins to
be bound to a matrix. For example, glutathione-S-transferase (GST) fusion
proteins or glutathione-S-transferase fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or
glutathione derivatized micro-titre plates, which are then combined with
the test compound or the test compound and either the non-adsorbed target
protein or PRCP, and the mixture incubated under conditions conducive to
complex formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtitre plate wells are washed to
remove any unbound components and complex formation is measured either
directly or indirectly, for example, as described above. Alternatively,
the complexes can be dissociated from the matrix, and the level of
binding or activity of PRCP can be determined using standard techniques.

[0159]Other techniques for immobilizing proteins on matrices can also be
used in the screening assays of the invention. For example, either PRCP
or its target molecule can be immobilized utilizing conjugation of biotin
and streptavidin. Biotinylated polypeptide of the invention or target
molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using
techniques well known in the art (e.g., biotinylation kit, Pierce
Chemicals; Rockford, Ill.), and immobilized in the wells of
streptavidin-coated plates (Pierce Chemical). Alternatively, antibodies
reactive with PRCP or target molecules but which do not interfere with
binding of the polypeptide of the invention to its target molecule can be
derivatized to the wells of the plate, and unbound target or polypeptide
of the invention trapped in the wells by antibody conjugation. Methods
for detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes using
antibodies reactive with PRCP or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with PRCP or target molecule.

[0160]Another technique for drug screening which may be used provides for
high throughput screening of compounds having suitable binding affinity
to the protein of interest as described in published PCT application
WO84/03564. In this method, large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic pins or
some other surface. The test compounds are reacted with PRCP, or
fragments thereof, and washed. Bound PRCP is then detected by methods
well known in the art. Purified PRCP can also be coated directly onto
plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the
peptide and immobilize it on a solid support.

[0161]In another embodiment, one may use competitive drug screening assays
in which neutralizing antibodies capable of binding PRCP specifically
compete with a testcompound for binding PRCP. In this manner, antibodies
can be used to detect the presence of any peptide which shares one or
more antigenic determinants with PRCP.

[0162]The screening assay can also involve monitoring the expression of
PRCP. For example, regulators of expression of PRCP can be identified in
a method in which a cell is contacted with a candidate compound and the
expression of PRCP protein or mRNA in the cell is determined. The level
of expression of PRCP protein or mRNA the presence of the candidate
compound is compared to the level of expression of PRCP protein or mRNA
in the absence of the candidate compound. The candidate compound can then
be identified as a regulator of expression of PRCP based on this
comparison. For example, when expression of PRCP protein or mRNA protein
is greater (statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of PRCP protein or mRNA expression.
Alternatively, when expression of PRCP protein or mRNA is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified as an
inhibitor of PRCP protein or mRNA expression. The level of PRCP protein
or mRNA expression in the cells can be determined by methods described
below.

Binding Assays

[0163]For binding assays, the test compound is preferably a small molecule
which binds to and occupies the active site of PRCP polypeptide, thereby
making the ligand binding site inaccessible to substrate such that normal
biological activity is prevented. Examples of such small molecules
include, but are not limited to, small peptides or peptide-like
molecules. Potential ligands which bind to a polypeptide of the invention
include, but are not limited to, the natural ligands of known PRCP
proteases and analogues or derivatives thereof.

[0164]In binding assays, either the test compound or the PRCP polypeptide
can comprise a detectable label, such as a fluorescent, radioisotopic,
chemiluminescent, or enzymatic label, such as horseradish peroxidase,
alkaline phosphatase, or luciferase. Detection of a test compound which
is bound to PRCP polypeptide can then be accomplished, for example, by
direct counting of radioemmission, by scintillation counting, or by
determining conversion of an appropriate substrate to a detectable
product. Alternatively, binding of a test compound to a PRCP poly-peptide
can be determined without labeling either of the interactants. For
example, a microphysiometer can be used to detect binding of a test
compound with a PRCP polypeptide. A microphysiometer (e.g.,
Cytosensor®) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate can be
used as an indicator of the interaction between a test compound and PRCP
[Haseloff, (1988)].

[0165]Determining the ability of a test compound to bind to PRCP also can
be accomplished using a technology such as real-time Bimolecular
Interaction Analysis (BIA) [McConnell, (1992); Sjolander, (1991)]. BIA is
a technology for studying biospecific interactions in real time, without
labeling any of the interactants (e.g., BIAcore®). Changes in the
optical phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.

[0166]In yet another aspect of the invention, a PRCP-like polypeptide can
be used as a "bait protein" in a two-hybrid assay or three-hybrid assay
[Szabo, (1995); U.S. Pat. No. 5,283,317), to identify other proteins
which bind to or interact with PRCP and modulate its activity.

[0167]The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. For example, in one construct, polynucleotide encoding PRCP
can be fused to a polynucleotide encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct a DNA
sequence that encodes an unidentified protein ("prey" or "sample") can be
fused to a polynucleotide that codes for the activation domain of the
known transcription factor. If the "bait" and the "prey" proteins are
able to interact in vivo to form an protein-dependent complex, the
DNA-binding and activation domains of the transcription factor are
brought into close proximity. This proximity allows transcription of a
reporter gene (e.g., LacZ), which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can be detected, and cell colonies containing the
functional transcription factor can be isolated and used to obtain the
DNA sequence encoding the protein which interacts with PRCP.

[0168]It may be desirable to immobilize either the PRCP (or
polynucleotide) or the test compound to facilitate separation of the
bound form from unbound forms of one or both of the interactants, as well
as to accommodate automation of the assay. Thus, either the PRCP-like
polypeptide (or polynucleotide) or the test compound can be bound to a
solid support. Suitable solid supports include, but are not limited to,
glass or plastic slides, tissue culture plates, microtiter wells, tubes,
silicon chips, or particles such as beads (including, but not limited to,
latex, polystyrene, or glass beads). Any method known in the art can be
used to attach PRCP-like polypeptide (or polynucleotide) or test compound
to a solid support, including use of covalent and non-covalent linkages,
passive absorption, or pairs of binding moieties attached respectively to
the polypeptide (or polynucleotide) or test compound and the solid
support. Test compounds are preferably bound to the solid support in an
array, so that the location of individual test compounds can be tracked.
Binding of a test compound to PRCP (or a polynucleotide encoding for
PRCP) can be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtiter plates, test
tubes, and microcentrifuge tubes.

[0169]In one embodiment, PRCP is a fusion protein comprising a domain that
allows binding of PRCP to a solid support. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined with
the test compound or the test compound and the non-adsorbed PRCP; the
mixture is then incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtiter plate wells are washed to remove any
unbound components. Binding of the interactants can be determined either
directly or indirectly, as described above. Alternatively, the complexes
can be dissociated from the solid support before binding is determined.

[0170]Other techniques for immobilizing proteins or polynucleotides on a
solid support also can be used in the screening assays of the invention.
For example, either PRCP (or a polynucleotide encoding PRCP) or a test
compound can be immobilized utilizing conjugation of biotin and
streptavidin. Biotinylated PRCP (or a polynucleotide encoding
biotinylated PRCP) or test compounds can be prepared from biotin-NHS
(N-hydroxysuccinimide) using techniques well known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in
the wells of streptavidin-coated plates (Pierce Chemical). Alternatively,
antibodies which specifically bind to PRCP, polynucleotide, or a test
compound, but which do not interfere with a desired binding site, such as
the active site of PRCP, can be derivatized to the wells of the plate.
Unbound target or protein can be trapped in the wells by antibody
conjugation.

[0171]Methods for detecting such complexes, in addition to those described
above for the GST-immobilized complexes, include immunodetection of
complexes using antibodies which specifically bind to PRCP polypeptide or
test compound, enzyme-linked assays which rely on detecting an activity
of PRCP polypeptide, and SDS gel electrophoresis under non-reducing
conditions.

[0172]Screening for test compounds which bind to a PRCP polypeptide or
polynucleotide also can be carried out in an intact cell. Any cell which
comprises a PRCP polypeptide or polynucleotide can be used in a
cell-based assay system. A PRCP polynucleotide can be naturally occurring
in the cell or can be introduced using techniques such as those described
above. Binding of the test compound to PRCP or a polynucleotide encoding
PRCP is determined as described above.

Functional Assays

[0173]Test compounds can be tested for the ability to increase or decrease
PRCP activity of a PRCP polypeptide. The PRCP activity can be measured,
for example, using methods described in the specific examples, below.
PRCP activity can be measured after contacting either a purified PRCP or
an intact cell with a test compound. A test compound which decreases PRCP
activity by at least about 10, preferably about 50, more preferably about
75, 90, or 100% is identified as a potential agent for decreasing PRCP
activity. A test compound which increases PRCP activity by at least about
10, preferably about 50, more preferably about 75, 90, or 100% is
identified as a potential agent for increasing PRCP activity.

Gene Expression

[0174]In another embodiment, test compounds which increase or decrease
PRCP gene expression are identified. As used herein, the term "correlates
with expression of a polynucleotide" indicates that the detection of the
presence of nucleic acids, the same or related to a nucleic acid sequence
encoding PRCP, by northern analysis or realtime PCR is indicative of the
presence of nucleic acids encoding PRCP in a sample, and thereby
correlates with expression of the transcript from the polynucleotide
encoding PRCP. The term "microarray", as used herein, refers to an array
of distinct polynucleotides or oligonucleotides arrayed on a substrate,
such as paper, nylon or any other type of membrane, filter, chip, glass
slide, or any other suitable solid support. A PRCP polynucleotide is
contacted with a test compound, and the expression of an RNA or
polypeptide product of PRCP polynucleotide is determined. The level of
expression of appropriate mRNA or polypeptide in the presence of the test
compound is compared to the level of expression of mRNA or polypeptide in
the absence of the test compound. The test compound can then be
identified as a regulator of expression based on this comparison. For
example, when expression of mRNA or polypeptide is greater in the
presence of the test compound than in its absence, the test compound is
identified as a stimulator or enhancer of the mRNA or polypeptide
expression. Alternatively, when expression of the mRNA or polypeptide is
less in the presence of the test compound than in its absence, the test
compound is identified as an inhibitor of the mRNA or polypeptide
expression.

[0175]The level of PRCP mRNA or polypeptide expression in the cells can be
determined by methods well known in the art for detecting mRNA or
polypeptide. Either qualitative or quantitative methods can be used. The
presence of polypeptide products of PRCP polynucleotide can be
deter-mined, for example, using a variety of techniques known in the art,
including immunochemical methods such as radioimmunoassay, Western
blotting, and immunohistochemistry. Alternatively, polypeptide synthesis
can be determined in vivo, in a cell culture, or in an in vitro
translation system by detecting incorporation of labelled amino acids
into PRCP.

[0176]Such screening can be carried out either in a cell-free assay system
or in an intact cell. Any cell which expresses PRCP polynucleotide can be
used in a cell-based assay system. The PRCP polynucleotide can be
naturally occurring in the cell or can be introduced using techniques
such as those described above. Either a primary culture or an established
cell line can be used.

Test Compounds

[0177]Suitable test compounds for use in the screening assays of the
invention can be obtained from any suitable source, e.g., conventional
compound libraries. The test compounds can also be obtained using any of
the numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection. The
biological library approach is limited to peptide libraries, while the
other four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds [Lam, (1997)]. Examples of methods
for the synthesis of molecular libraries can be found in the art.
Libraries of compounds may be presented in solution or on beads,
bacteria, spores, plasmids or phage.

Modeling of Regulators

[0178]Computer modeling and searching technologies permit identification
of compounds, or the improvement of already identified compounds, that
can modulate PRCP expression or activity. Having identified such a
compound or composition, the active sites or regions are identified. Such
sites might typically be the enzymatic active site, regulator binding
sites, or ligand binding sites. The active site can be identified using
methods known in the art including, for example, from the amino acid
sequences of peptides, from the nucleotide sequences of nucleic acids, or
from study of complexes of the relevant compound or composition with its
natural ligand. In the latter case, chemical or X-ray crystallographic
methods can be used to find the active site by finding where on the
factor the complexed ligand is found.

[0179]Next, the three dimensional geometric structure of the active site
is determined. This can be done by known methods, including X-ray
crystallography, which can determine a complete molecular structure. On
the other hand, solid or liquid phase NMR can be used to determine
certain intramolecular distances. Any other experimental method of
structure determination can be used to obtain partial or complete
geometric structures. The geometric structures may be measured with a
complexed ligand, natural or artificial, which may increase the accuracy
of the active site structure determined.

[0180]If an incomplete or insufficiently accurate structure is determined,
the methods of computer based numerical modeling can be used to complete
the structure or improve its accuracy. Any recognized modeling method may
be used, including parameterized models specific to particular
biopolymers such as proteins or nucleic acids, molecular dynamics models
based on computing molecular motions, statistical mechanics models based
on thermal ensembles, or combined models. For most types of models,
standard molecular force fields, representing the forces between
constituent atoms and groups, are necessary, and can be selected from
force fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete and more
accurate structures computed by these modeling methods.

[0181]Finally, having determined the structure of the active site, either
experimentally, by modeling, or by a combination, candidate modulating
compounds can be identified by searching databases containing compounds
along with information on their molecular structure. Such a search seeks
compounds having structures that match the determined active site
structure and that interact with the groups defining the active site.
Such a search can be manual, but is preferably computer assisted. These
compounds found from this search are potential PRCP modulating compounds.
Alternatively, these methods can be used to identify improved modulating
compounds from an already known modulating compound or ligand. The
composition of the known compound can be modified and the structural
effects of modification can be determined using the experimental and
computer modeling methods described above applied to the new composition.
The altered structure is then compared to the active site structure of
the compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by varying side
groups, can be quickly evaluated to obtain modified modulating compounds
or ligands of improved specificity or activity.

Therapeutic Indications and Methods

[0182]It was found by the present applicant that PRCP is expressed in
various human tissues.

Neurology

[0183]CNS disorders include disorders of the central nervous system as
well as disorders of the peripheral nervous system.

[0190]Heart failure is defined as a pathophysiological state in which an
abnormality of cardiac function is responsible for the failure of the
heart to pump blood at a rate commensurate with the requirement of the
metabolizing tissue. It includes all forms of pumping failures such as
high-output and low-output, acute and chronic, right-sided or left-sided,
systolic or diastolic, independent of the underlying cause.

[0191]Myocardial infarction (MI) is generally caused by an abrupt decrease
in coronary blood flow that follows a thrombotic occlusion of a coronary
artery previously narrowed by arteriosclerosis. MI prophylaxis (primary
and secondary prevention) is included as well as the acute treatment of
MI and the prevention of complications.

[0192]Ischemic diseases are conditions in which the coronary flow is
restricted resulting in a perfusion which is inadequate to meet the
myocardial requirement for oxygen. This group of diseases includes stable
angina, unstable angina and asymptomatic ischemia.

[0194]Hypertensive vascular diseases include primary as well as all kinds
of secondary arterial hypertension, renal, endocrine, neurogenic, others.
The genes may be used as drug targets for the treatment of hypertension
as well as for the prevention of all complications arising from
cardiovascular diseases.

[0196]Atherosclerosis is a cardiovascular disease in which the vessel wall
is remodeled, compromising the lumen of the vessel. The atherosclerotic
remodeling process involves accumulation of cells, both smooth muscle
cells and monocyte/macrophage inflammatory cells, in the intima of the
vessel wall. These cells take up lipid, likely from the circulation, to
form a mature atherosclerotic lesion. Although the formation of these
lesions is a chronic process, occurring over decades of an adult human
life, the majority of the morbidity associated with atherosclerosis
occurs when a lesion ruptures, releasing thrombogenic debris that rapidly
occludes the artery. When such an acute event occurs in the coronary
artery, myocardial infarction can ensue, and in the worst case, can
result in death.

[0197]The formation of the atherosclerotic lesion can be considered to
occur in five overlapping stages such as migration, lipid accumulation,
recruitment of inflammatory cells, proliferation of vascular smooth
muscle cells, and extracellular matrix deposition. Each of these
processes can be shown to occur in man and in animal models of
atherosclerosis, but the relative contribution of each to the pathology
and clinical significance of the lesion is unclear.

[0198]Thus, a need exists for therapeutic methods and agents to treat
cardiovascular pathologies, such as atherosclerosis and other conditions
related to coronary artery disease.

[0199]Cardiovascular diseases include but are not limited to disorders of
the heart and the vascular system like congestive heart failure,
myocardial infarction, ischemic diseases of the heart, all kinds of
atrial and ventricular arrhythmias, hypertensive vascular diseases,
peripheral vascular diseases, and atherosclerosis.

[0200]Too high or too low levels of fats in the bloodstream, especially
cholesterol, can cause long-term problems. The risk to develop
atherosclerosis and coronary artery or carotid artery disease (and thus
the risk of having a heart attack or stroke) increases with the total
cholesterol level increasing. Nevertheless, extremely low cholesterol
levels may not be healthy. Examples of disorders of lipid metabolism are
hyperlipidemia (abnormally high levels of fats (cholesterol,
triglycerides, or both) in the blood, may be caused by family history of
hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to
high alcohol consumption, cigarette smoking, poorly controlled diabetes,
and an underactive thyroid gland), hereditary hyperlipidemias (type I
hyperlipoproteinemia (familial hyperchylomicronemia), type II
hyperlipoproteinemia (familial hypercholesterolemia), type DI
hyperlipoproteinemia, type IV hyperlipoproteinemia, or type V
hyperlipoproteinemia), hypolipo-proteinemia, lipidoses (caused by
abnormalities in the enzymes that metabolize fats), Gaucher's disease,
Niemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous
xantho-matosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.

[0201]Kidney disorders may lead to hypertension or hypotension. Examples
for kidney problems possibly leading to hypertension are renal artery
stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic
kidney disease, injury to the kidney, or radiation therapy affecting the
kidney. Excessive urination may lead to hypotension.

[0203]The human PRCP is highly expressed in liver tissues: fetal liver,
liver, liver liver cirrhosis, liver tumor. Expression in liver tissues
demonstrates that the human PRCP or mRNA can be utilized to diagnose of
dyslipidemia disorders as an cardiovascular disorder. Additionally the
activity of the human PRCP can be modulated to treat but not limited to
dyslipidemia disorders.

[0204]The human PRCP is highly expressed in kidney tissues: fetal kidney,
kidney, kidney, kidney tumor, HEK 293 cells. Expression in kidney tissues
demonstrates that the human PRCP or mRNA can be utilized to diagnose of
blood pressure disorders as an cardiovascular disorder. Additionally the
activity of the human PRCP can be modulated to treat but not limited to
blood pressure disorders as hypertension or hypotension.

Hematological Disorders

[0205]Hematological disorders comprise diseases of the blood and all its
constituents as well as diseases of organs and tissues involved in the
generation or degradation of all the constituents of the blood. They
include but are not limited to 1) Anemias, 2) Myeloproliferative
Disorders, 3) Hemorrhagic Disorders, 4) Leukopenia, 5) Eosinophilic
Disorders, 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9)
Disorders of the Spleen in the course of hematological disorders.
Disorders according to 1) include, but are not limited to anemias due to
defective or deficient hem synthesis, deficient erythropoiesis. Disorders
according to 2) include, but are not limited to polycythemia vera,
tumor-associated erythrocytosis, myelofibrosis, thrombocythemia.
Disorders according to 3) include, but are not limited to vasculitis,
thrombocytopenia, heparin-induced thrombocytopenia, thrombotic
thrombocytopenic purpura, hemolytic-uremic syndrome, hereditary and
acquired disorders of platelet function, hereditary coagulation
disorders. Disorders according to 4) include, but are not limited to
neutropenia, lymphocytopenia. Disorders according to 5) include, but are
not limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.
Disorders according to 6) include, but are not limited to acute myeloic
leukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia,
chronic lymphocytic leukemia, myelodysplastic syndrome. Disorders
according to 7) include, but are not limited to Hodgkin's disease,
non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoides cutaneous
T-cell lymphoma. Disorders according to 8) include, but are not limited
to multiple myeloma, macroglobulinemia, heavy chain diseases. In
extension of the preceding idiopathic thrombocytopenic purpura, iron
deficiency anemia, megaloblastic anemia (vitamin B12 deficiency),
aplastic anemia, thalassemia, malignant lymphoma bone marrow invasion,
malignant lymphoma skin invasion, hemolytic uremic syndrome, giant
platelet disease are considered to be hematological diseases too.

[0207]Cancer disorders within the scope of this definition comprise any
disease of an organ or tissue in mammals characterized by poorly
controlled or uncontrolled multiplication of normal or abnormal cells in
that tissue and its effect on the body as a whole. Cancer diseases within
the scope of the definition comprise benign neoplasms, dysplasias,
hyperplasias as well as neoplasms showing metastatic growth or any other
transformations like e.g. leukoplakias which often precede a breakout of
cancer. Cells and tissues are cancerous when they grow more rapidly than
normal cells, displacing or spreading into the surrounding healthy tissue
or any other tissues of the body described as metastatic growth, assume
abnormal shapes and sizes, show changes in their nucleocytoplasmatic
ratio, nuclear polychromasia, and finally may cease. Cancerous cells and
tissues may affect the body as a whole when causing paraneoplastic
syndromes or if cancer occurs within a vital organ or tissue, normal
function will be impaired or halted, with possible fatal results. The
ultimate involvement of a vital organ by cancer, either primary or
metastatic, may lead to the death of the mammal affected. Cancer tends to
spread, and the extent of its spread is usually related to an
individual's chances of surviving the disease. Cancers are generally said
to be in one of three stages of growth: early, or localized, when a tumor
is still confined to the tissue of origin, or primary site; direct
extension, where cancer cells from the tumour have invaded adjacent
tissue or have spread only to regional lymph nodes; or metastasis, in
which cancer cells have migrated to distant parts of the body from the
primary site, via the blood or lymph systems, and have established
secondary sites of infection. Cancer is said to be malignant because of
its tendency to cause death if not treated. Benign tumors usually do not
cause death, although they may if they interfere with a normal body
function by virtue of their location, size, or paraneoplastic side
effects. Hence benign tumors fall under the definition of cancer within
the scope of this definition as well. In general, cancer cells divide at
a higher rate than do normal cells, but the distinction between the
growth of cancerous and normal tissues is not so much the rapidity of
cell division in the former as it is the partial or complete loss of
growth restraint in cancer cells and their failure to differentiate into
a useful, limited tissue of the type that characterizes the functional
equilibrium of growth of normal tissue. Cancer tissues may express
certain molecular receptors and probably are influenced by the host's
susceptibility and immunity and it is known that certain cancers of the
breast and prostate, for example, are considered dependent on specific
hormones for their existence. The term "cancer" under the scope of the
definition is not limited to simple benign neoplasia but comprises any
other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3)
Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors of
nerve tissues including the brain, 6) cancer of skin cells. Cancer
according to 1) occurs in epithelial tissues, which cover the outer body
(the skin) and line mucous membranes and the inner cavitary structures of
organs e.g. such as the breast, lung, the respiratory and
gastrointestinal tracts, the endocrine glands, and the genitourinary
system. Ductal or glandular elements may persist in epithelial tumors, as
in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric
adenocarcinoma, uterine adenocarci-noma. Cancers of the pavement-cell
epithelium of the skin and of certain mucous membranes, such as e.g.
cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, or
penis, may be termed epidermoid or squamous-cell carcinomas of the
respective tissues and are in the scope of the definition of cancer as
well. Cancer according to 2) develops in connective tissues, including
fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and
cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma,
synovial sarcoma. Cancer according to 3) is cancer that develops in both
epithelial and connective tissue. Cancer disease within the scope of this
definition may be primary or secondary, whereby primary indicates that
the cancer originated in the tissue where it is found rather than was
established as a secondary site through metastasis from another lesion.
Cancers and tumor diseases within the scope of this definition may be
benign or malign and may affect all anatomical structures of the body of
a mammal. By example but not limited to they comprise cancers and tumor
diseases of I) the bone marrow and bone marrow derived cells (leukemias),
II) the endocrine and exocrine glands like e.g. thyroid, parathyroid,
pituitary, adrenal glands, salivary glands, pancreas III) the breast,
like e.g. benign or malignant tumors in the mammary glands of either a
male or a female, the mammary ducts, adenocarcinoma, medullary carcinoma,
comedo carcinoma, Paget's disease of the nipple, inflammatory carcinoma
of the young woman, IV) the lung, V) the stomach, VI) the liver and
spleen, VII) the small intestine, VIII) the colon, IX) the bone and its
supportive and connective tissues like malignant or benign bone tumour,
e.g. malignant osteogenic sarcoma, benign osteoma, cartilage tumors; like
malignant chon-drosarcoma or benign chondroma; bone marrow tumors like
malignant myeloma or benign eosinophilic granuloma, as well as metastatic
tumors from bone tissues at other locations of the body; X) the mouth,
throat, larynx, and the esophagus, XI) the urinary bladder and the
internal and external organs and structures of the urogenital system of
male and female like ovaries, uterus, cervix of the uterus, testes, and
prostate gland, XII) the prostate, XIII) the pancreas, like ductal
carcinoma of the pancreas; XIV) the lymphatic tissue like lymphomas and
other tumors of lymphoid origin, XV) the skin, XVI) cancers and tumor
diseases of all anatomical structures belonging to the respiration and
respiratory systems including thoracal muscles and linings, XVII) primary
or secondary cancer of the lymph nodes XVIII) the tongue and of the bony
structures of the hard palate or sinuses, XVIV) the mouth, cheeks, neck
and salivary glands, XX) the blood vessels including the heart and their
linings, XXI) the smooth or skeletal muscles and their ligaments and
linings, XXII) the peripheral, the autonomous, the central nervous system
including the cerebellum, XXIII) the adipose tissue.

[0210]The regulatory method of the invention involves contacting a cell
with an agent that modulates one or more of the activities of PRCP. An
agent that modulates activity can be an agent as described herein, such
as a nucleic acid or a protein, a naturally-occurring cognate ligand of
the polypeptide, a peptide, a peptidomimetic, or any small molecule. In
one embodiment, the agent stimulates one or more of the biological
activities of PRCP. Examples of such stimulatory agents include the
active PRCP and nucleic acid molecules encoding a portion of PRCP. In
another embodiment, the agent inhibits one or more of the biological
activities of PRCP. Examples of such inhibitory agents include antisense
nucleic acid molecules and antibodies. These regulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g, by administering the agent to a subject). As
such, the present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by unwanted expression
or activity of PRCP or a protein in the PRCP signaling pathway. In one
embodiment, the method involves administering an agent like any agent
identified or being identifiable by a screening assay as described
herein, or combination of such agents that modulate say upregulate or
downregulate the expression or activity of PRCP or of any protein in the
PRCP signaling pathway. In another embodiment, the method involves
administering a regulator of PRCP as therapy to compensate for reduced or
undesirably low expression or activity of PRCP or a protein in the PRCP
signaling pathway.

[0211]Stimulation of activity or expression of PRCP is desirable in
situations in which enzymatic activity or expression is abnormally low
and in which increased activity is likely to have a beneficial effect.
Conversely, inhibition of enzymatic activity or expression of PRCP is
desirable in situations in which activity or expression of PRCP is
abnormally high and in which decreasing its activity is likely to have a
beneficial effect.

[0212]This invention is further illustrated by the following examples
which should not be construed as limiting. The contents of all
references, patents and published patent applications cited throughout
this application are hereby incorporated by reference.

Pharmaceutical Compositions

[0213]This invention further pertains to novel agents identified by the
above-described screening assays and uses thereof for treatments as
described herein.

[0214]The nucleic acid molecules, polypeptides, and antibodies (also
referred to herein as "active compounds") of the invention can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier.
As used herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying
agents, and the like, compa-tible with pharmaceutical administration. The
use of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the compositions
is contemplated. Supplementary active compounds can also be incorporated
into the compositions.

[0215]The invention includes pharmaceutical compositions comprising a
regulator of PRCP expression or activity (and/or a regulator of the
activity or expression of a protein in the PRCP signaling pathway) as
well as methods for preparing such compositions by combining one or more
such regulators and a pharmaceutically acceptable carrier. Also within
the invention are pharmaceutical compositions comprising a regulator
identified using the screening assays of the invention packaged with
instructions for use. For regulators that are antagonists of PRCP
activity or which reduce PRCP expression, the instructions would specify
use of the pharmaceutical composition for treatment of cardiovascular
diseases, hematological diseases, neurological diseases and cancer. For
regulators that are agonists of PRCP activity or increase PRCP
expression, the instructions would specify use of the pharmaceutical
composition for treatment of cardiovascular diseases, hematological
diseases, neurological diseases and cancer.

[0216]An inhibitor of PRCP may be produced using methods which are
generally known in the art. In particular, purified PRCP may be used to
produce antibodies or to screen libraries of pharmaceutical agents to
identify those which specifically bind PRCP. Antibodies to PRCP may also
be generated using methods that are well known in the art. Such
antibodies may include, but are not limited to, polyclonal, monoclonal,
chimeric, single chain antibodies, Fab fragments, and fragments produced
by a Fab expression library. Neutralizing antibodies like those which
inhibit dimer formation are especially preferred for therapeutic use.

[0217]In another embodiment of the invention, the polynucleotides encoding
PRCP, or any fragment or complement thereof, may be used for therapeutic
purposes. In one aspect, the complement of the polynucleotide encoding
PRCP may be used in situations in which it would be desirable to block
the transcription of the mRNA. In particular, cells may be transformed
with sequences complementary to polynucleotides encoding PRCP. Thus,
complementary molecules or fragments may be used to modulate PRCP
activity, or to achieve regulation of gene function. Such technology is
now well known in the art, and sense or antisense oligonucleotides or
larger fragments can be designed from various locations along the coding
or control regions of sequences encoding PRCP.

[0218]Expression vectors derived from retroviruses, adenoviruses, or
herpes or vaccinia viruses, or from various bacterial plasmids, may be
used for delivery of nucleotide sequences to the targeted organ, tissue,
or cell population. Methods which are well known to those skilled in the
art can be used to construct vectors which will express nucleic acid
sequence complementary to the polynucleotides of the gene encoding PRCP.
These techniques are described, for example, in [Scott and Smith (1990)].

[0219]Any of the therapeutic methods described above may be applied to any
subject in need of such therapy, including, for example, mammals such as
dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

[0220]An additional embodiment of the invention relates to the
administration of a pharmaceutical composition containing PRCP in
conjunction with a pharmaceutically acceptable carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of PRCP, antibodies to PRCP, and mimetics, agonists, antagonists,
or inhibitors of PRCP. The compositions may be administered alone or in
combination with at least one other agent, such as a stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier including, but not limited to, saline, buffered
saline, dextrose, and water. The compositions may be administered to a
patient alone, or in combination with other agents, drugs or hormones.

[0221]A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes
of administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions used
for parenteral, intradermal, or subcutaneous application can include the
following components: a sterile diluent such as water for injection,
saline solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or plastic.

[0222]Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water, Cremophor
EM® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In
all cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi. The
carrier can be a solvent or dispersion medium containing, for example,
water, ethanol, a pharmaceutically acceptable polyol like glycerol,
propylene glycol, liquid polyetheylene glycol, and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many
cases, it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays
absorption, for example, aluminum monostearate and gelatin. Sterile
injectable solutions can be prepared by incorporating the active compound
(e.g., a polypeptide or antibody) in the required amount in an
appropriate solvent with one or a combination of ingredients enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into a
sterile vehicle which contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and freeze-drying
which yields a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution thereof.

[0223]Oral compositions generally include an inert diluent or an edible
carrier. They can be enclosed in gelatin capsules or compressed into
tablets. For the purpose of oral therapeutic administration, the active
compound can be incorporated with excipients and used in the form of
tablets, troches, or capsules. Oral compositions can also be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed.

[0224]Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such
as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or corn starch; a lubricant such as magnesium stearate or
sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.

[0225]For administration by inhalation, the compounds are delivered in the
form of an aerosol spray from a pressurized container or dispenser which
contains a suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer.

[0226]Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art, and include, for example,
for transmucosal administration, detergents, bile salts, and fusidic acid
derivatives. Transmucosal administration can be accomplished through the
use of nasal sprays or suppositories. For transdermal administration, the
active compounds are formulated into ointments, salves, gels, or creams
as generally known in the art.

[0227]The compounds can also be prepared in the form of suppositories
(e.g., with conventional suppository bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.

[0228]In one embodiment, the active compounds are prepared with carriers
that will protect the compound against rapid elimination from the body,
such as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled in the
art. The materials can also be obtained commercially from Alza
Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions
(including liposomes targeted to infected cells with monoclonal
antibodies to viral antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods known to
those skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.

[0229]It is especially advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association with
the required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in the
art of compounding such an active compound for the treatment of
individuals.

[0230]The pharmaceutical compositions can be included in a container,
pack, or dispenser together with instructions for administration. For
pharmaceutical compositions which include an antagonist of PRCP activity,
a compound which reduces expression of PRCP, or a compound which reduces
expression or activity of a protein in the PRCP signaling pathway or any
combination thereof, the instructions for administration will specify use
of the composition for cardiovascular diseases, hematological diseases,
neurological diseases and cancer. For pharmaceutical compositions which
include an agonist of PRCP activity, a compound which increases
expression of PRCP, or a compound which increases expression or activity
of a protein in the PRCP signaling pathway or any combination thereof,
the instructions for administration will specify use of the composition
for cardiovascular diseases, hematological diseases, neurological
diseases and cancer.

Diagnostics

[0231]In another embodiment, antibodies which specifically bind PRCP may
be used for the diagnosis of disorders characterized by the expression of
PRCP, or in assays to monitor patients being treated with PRCP or
agonists, antagonists, and inhibitors of PRCP. Antibodies useful for
diagnostic purposes may be prepared in the same manner as those described
above for therapeutics. Diagnostic assays for PRCP include methods which
utilize the antibody and a label to detect PRCP in human body fluids or
in extracts of cells or tissues. The antibodies may be used with or
without modification, and may be labeled by covalent or non-covalent
joining with a reporter molecule. A wide variety of reporter molecules,
several of which are described above, are known in the art and may be
used.

[0232]A variety of protocols for measuring PRCP, including ELISAs, RIAs,
and FACS, are known in the art and provide a basis for diagnosing altered
or abnormal levels of PRCP expression. Normal or standard values for PRCP
expression are established by combining body fluids or cell extracts
taken from normal mammalian subjects, preferably human, with antibody to
PRCP under conditions suitable for complex formation. The amount of
standard complex formation may be quantified by various methods,
preferably by photometric means. Quantities of PRCP expressed in subject
samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters
for diagnosing disease.

[0233]In another embodiment of the invention, the polynucleotides encoding
PRCP may be used for diagnostic purposes. The polynucleotides which may
be used include oligonucleotide sequences, complementary RNA and DNA
molecules, and PNAs. The polynucleotides may be used to detect and
quantitate gene expression in biopsied tissues in which expression of
PRCP may be correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of PRCP, and
to monitor regulation of PRCP levels during therapeutic intervention.

[0234]Polynucleotide sequences encoding PRCP may be used for the diagnosis
of cardiovascular diseases, hematological diseases, neurological diseases
and cancer associated with expression of PRCP. The polynucleotide
sequences encoding PRCP may be used in Southern, Northern, or dot-blot
analysis, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or
tissues from patient biopsies to detect altered PRCP expression. Such
qualitative or quantitative methods are well known in the art.

[0235]In a particular aspect, the nucleotide sequences encoding PRCP may
be useful in assays that detect the presence of associated disorders,
particularly those mentioned above. The nucleotide sequences encoding
PRCP may be labelled by standard methods and added to a fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a suitable incubation period, the sample
is washed and the signal is quantitated and compared with a standard
value. If the amount of signal in the patient sample is significantly
altered from that of a comparable control sample, the nucleotide
sequences have hybridized with nucleotide sequences in the sample, and
the presence of altered levels of nucleotide sequences encoding PRCP in
the sample indicates the presence of the associated disorder. Such assays
may also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in animal studies, in clinical trials, or in monitoring
the treatment of an individual patient.

[0236]In order to provide a basis for the diagnosis of cardiovascular
diseases, hematological diseases, neurological diseases and cancer
associated with expression of PRCP, a normal or standard profile for
expression is established. This may be accomplished by combining body
fluids or cell extracts taken from normal subjects, either animal or
human, with a sequence, or a fragment thereof, encoding PRCP, under
conditions suitable for hybridization or amplification. Standard
hybridiza-tion may be quantified by comparing the values obtained from
normal subjects with values from an experiment in which a known amount of
a substantially purified polynucleotide is used. Standard values obtained
from normal samples may be compared with values obtained from samples
from patients who are symptomatic for a disorder. Deviation from standard
values is used to establish the presence of a disorder.

Determination of a Therapeutically Effective Dose

[0237]The determination of a therapeutically effective dose is well within
the capability of those skilled in the art. A therapeutically effective
dose refers to that amount of active ingredient which increases or
decreases PRCP activity relative to PRCP activity which occurs in the
absence of the therapeutically effective dose. For any compound, the
therapeutically effective dose can be estimated initially either in cell
culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
The animal model also can be used to determine the appropriate
concentration range and route of administration. Such information can
then be used to determine useful doses and routes for administration in
humans.

[0238]Therapeutic efficacy and toxicity, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population), can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals. The
dose ratio of toxic to therapeutic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The
data obtained from cell culture assays and animal studies is used in
formulating a range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating concentrations
that include the ED50 with little or no toxicity. The dosage varies
within this range depending upon the dosage form employed, sensitivity of
the patient, and the route of administration. The exact dosage will be
determined by the practitioner, in light of factors related to the
subject that requires treatment. Dosage and administration are adjusted
to provide sufficient levels of the active ingredient or to maintain the
desired effect. Factors which can be taken into account include the
severity of the disease state, general health of the subject, age,
weight, and gender of the subject, diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical compositions
can be administered every 3 to 4 days, every week, or once every two
weeks depending on the half-life and clearance rate of the particular
formulation.

[0239]Normal dosage amounts can vary from 0.1 micrograms to 100,000
micrograms, up to a total dose of about 1 g, depending upon the route of
administration. Guidance as to particular dosages and methods of delivery
is provided in the literature and generally available to practitioners in
the art. Those skilled in the art will employ different formulations for
nucleotides than for proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular cells,
conditions, locations, etc. If the reagent is a single-chain antibody,
polynucleotides encoding the antibody can be constructed and introduced
into a cell either ex vivo or in vivo using well-established techniques
including, but not limited to, transferrin-polycation-mediated DNA
transfer, transfection with naked or encapsulated nucleic acids,
liposome-mediated cellular fusion, intra-cellular transportation of
DNA-coated latex beads, protoplast fusion, viral infection,
electro-poration, "gene gun", and DEAE- or calcium phosphate-mediated
transfection.

[0240]If the expression product is mRNA, the reagent is preferably an
antisense oligonucleotide or a ribozyme. Polynucleotides which express
antisense oligonucleotides or ribozymes can be intro-duced into cells by
a variety of methods, as described above. Preferably, a reagent reduces
expression of PRCP gene or the activity of PRCP by at least about 10,
preferably about 50, more preferably about 75, 90, or 100% relative to
the absence of the reagent. The effectiveness of the mechanism chosen to
decrease the level of expression of PRCP gene or the activity of PRCP can
be assessed using methods well known in the art, such as hybridization of
nucleotide probes to PRCP-specific mRNA, quantitative RT-PCR, immunologic
detection of PRCP, or measurement of PRCP activity.

[0241]In any of the embodiments described above, any of the pharmaceutical
compositions of the invention can be administered in combination with
other appropriate therapeutic agents. Selection of the appropriate agents
for use in combination therapy can be made by one of ordinary skill in
the art, according to conventional pharmaceutical principles. The
combination of therapeutic agents can act synergistically to effect the
treatment or prevention of the various disorders described above. Using
this approach, one may be able to achieve therapeutic efficacy with lower
dosages of each agent, thus reducing the potential for adverse side
effects. Any of the therapeutic methods described above can be applied to
any subject in need of such therapy, including, for example, mammals such
as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,
humans.

[0242]Nucleic acid molecules of the invention are those nucleic acid
molecules which are contained in a group of nucleic acid molecules
consisting of (i) nucleic acid molecules encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, (ii) nucleic acid
molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid
molecules having the sequence of SEQ ID NO: 1, (iv) nucleic acid
molecules the complementary strand of which hybridizes under stringent
conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v)
nucleic acid molecules the sequence of which differs from the sequence of
a nucleic acid molecule of (iii) due to the degeneracy of the genetic
code, wherein the polypeptide encoded by said nucleic acid molecule has
PRCP activity.

[0243]Polypeptides of the invention are those polypeptides which are
contained in a group of poly-peptides consisting of (i) polypeptides
having the sequence of SEQ ID NO: 2, (ii) polypeptides comprising the
sequence of SEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid
molecules of the invention and (iv) polypeptides which show at least 99%,
98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii),
wherein said purified polypeptide has PRCP activity.

[0244]An object of the invention is a method of screening for therapeutic
agents useful in the treatment of a disease comprised in a group of
diseases consisting of cardiovascular diseases, hematological diseases,
neurological diseases and cancer in a mammal comprising the steps of (i)
contacting a test compound with a PRCP polypeptide, (ii) detect binding
of said test compound to said PRCP polypeptide. E.g., compounds that bind
to the PRCP polypeptide are identified potential therapeutic agents for
such a disease.

[0245]Another object of the invention is a method of screening for
therapeutic agents useful in the treatment of a disease comprised in a
group of diseases consisting of cardiovascular diseases, hematological
diseases, neurological diseases and cancer in a mammal comprising the
steps of (i) determining the activity of a PRCP polypeptide at a certain
concentration of a test compound or in the absence of said test compound,
(ii) determining the activity of said polypeptide at a different
concentration of said test compound. E.g., compounds that lead to a
difference in the activity of the PRCP polypeptide in (i) and (ii) are
identified potential therapeutic agents for such a disease.

[0246]Another object of the invention is a method of screening for
therapeutic agents useful in the treatment of a disease comprised in a
group of diseases consisting of cardiovascular diseases, hematological
diseases, neurological diseases and cancer in a mammal comprising the
steps of (i) determining the activity of a PRCP polypeptide at a certain
concentration of a test compound, (ii) determining the activity of a PRCP
polypeptide at the presence of a compound known to be a regulator of a
PRCP polypeptide. E.g., compounds that show similar effects on the
activity of the PRCP polypeptide in (i) as compared to compounds used in
(ii) are identified potential therapeutic agents for such a disease.

[0247]Other objects of the invention are methods of the above, wherein the
step of contacting is in or at the surface of a cell.

[0248]Other objects of the invention are methods of the above, wherein the
cell is in vitro.

[0249]Other objects of the invention are methods of the above, wherein the
step of contacting is in a cell-free system.

[0250]Other objects of the invention are methods of the above, wherein the
polypeptide is coupled to a detectable label.

[0251]Other objects of the invention are methods of the above, wherein the
compound is coupled to a detectable label.

[0252]Other objects of the invention are methods of the above, wherein the
test compound displaces a ligand which is first bound to the polypeptide.

[0253]Other objects of the invention are methods of the above, wherein the
polypeptide is attached to a solid support.

[0254]Other objects of the invention are methods of the above, wherein the
compound is attached to a solid support.

[0255]Another object of the invention is a method of screening for
therapeutic agents useful in the treatment of a disease comprised in a
group of diseases consisting of cardiovascular diseases, hematological
diseases, neurological diseases and cancer in a mammal comprising the
steps of (i) contacting a test compound with a PRCP polynucleotide, (ii)
detect binding of said test compound to said PRCP polynucleotide.
Compounds that, e.g., bind to the PRCP polynucleotide are potential
therapeutic agents for the treatment of such diseases.

[0256]Another object of the invention is the method of the above, wherein
the nucleic acid molecule is RNA.

[0257]Another object of the invention is a method of the above, wherein
the contacting step is in or at the surface of a cell.

[0258]Another object of the invention is a method of the above, wherein
the contacting step is in a cell-free system.

[0259]Another object of the invention is a method of the above, wherein
the polynucleotide is coupled to a detectable label.

[0260]Another object of the invention is a method of the above, wherein
the test compound is coupled to a detectable label.

[0261]Another object of the invention is a method of diagnosing a disease
comprised in a group of diseases consisting of cardiovascular diseases,
hematological diseases, neurological diseases and cancer in a mammal
comprising the steps of (i) determining the amount of a PRCP
polynucleotide in a sample taken from said mammal, (ii) determining the
amount of PRCP polynucleotide in healthy and/or diseased mammal. A
disease is diagnosed, e.g., if there is a substantial similarity in the
amount of PRCP polynucleotide in said test mammal as compared to a
diseased mammal.

[0262]Another object of the invention is a pharmaceutical composition for
the treatment of a disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal comprising a therapeutic agent which binds to a
PRCP polypeptide.

[0263]Another object of the invention is a pharmaceutical composition for
the treatment of a disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal comprising a therapeutic agent which regulates the
activity of a PRCP polypeptide.

[0264]Another object of the invention is a pharmaceutical composition for
the treatment of a disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal comprising a therapeutic agent which regulates the
activity of a PRCP polypeptide, wherein said therapeutic agent is (i) a
small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide,
(iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.

[0265]Another object of the invention is a pharmaceutical composition for
the treatment of a disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal comprising a PRCP polynucleotide.

[0266]Another object of the invention is a pharmaceutical composition for
the treatment of a disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer in a mammal comprising a PRCP polypeptide.

[0267]Another object of the invention is the use of regulators of a PRCP
for the preparation of a pharmaceutical composition for the treatment of
a disease comprised in a group of diseases consisting of cardiovascular
diseases, hematological diseases, neurological diseases and cancer in a
mammal.

[0268]Another object of the invention is a method for the preparation of a
pharmaceutical composition useful for the treatment of a disease
comprised in a group of diseases consisting of cardiovascular diseases,
hematological diseases, neurological diseases and cancer in a mammal
comprising the steps of (i) identifying a regulator of PRCP, (ii)
determining whether said regulator ameliorates the symptoms of a disease
comprised in a group of diseases consisting of cardiovascular diseases,
hematological diseases, neurological diseases and cancer in a mammal; and
(iii) combining of said regulator with an acceptable pharmaceutical
carrier.

[0269]Another object of the invention is the use of a regulator of PRCP
for the regulation of PRCP activity in a mammal having a disease
comprised in a group of diseases consisting of cardiovascular diseases,
hematological diseases, neurological diseases and cancer.

[0270]The uses, methods or compositions of the invention are useful for
each single disease comprised in a group of diseases consisting of
cardiovascular diseases, hematological diseases, neurological diseases
and cancer.

[0271]The expression of human PRCP in cardiovascular diseases and
hematological diseases related tissues (as described above) suggests a
particular--but not limited to--utilization of PRCP for diagnosis and
modulation of cardiovascular diseases and hematological diseases.
Furthermore the above described expression suggest a--but not limited
to--utilization of PRCP to neurological diseases and cancer.

[0272]The examples below are provided to illustrate the subject invention.
These examples are provided by way of illustration and are not included
for the purpose of limiting the invention.

EXAMPLES

Example 1

Search for Homologous Sequences in Public Sequence Data Bases

[0273]The degree of homology can readily be calculated by known methods.
Preferred methods to determine homology are designed to give the largest
match between the sequences tested. Methods to determine homology are
codified in publicly available computer programs such as BestFit, BLASTP,
BLASTN, and FASTA. The BLAST programs are publicly available from NCBI
and other sources in the internet.

[0276]Total cellular RNA was isolated from cells by one of two standard
methods: 1) guanidine isothio-cyanate/Cesium chloride density gradient
centrifugation [Kellogg, (1990)]; or with the Tri-Reagent protocol
according to the manufacturer's specifications (Molecular Research
Center, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagent
protocol was treated with DNAse I to remove genomic DNA contamination.

[0277]For relative quantitation of the mRNA distribution of PRCP, total
RNA from each cell or tissue source was first reverse transcribed. 85
μg of total RNA was reverse transcribed using 1 μmole random
hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen,
Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in
a final volume of 680 μl. The first strand synthesis buffer and
Omniscript reverse transcriptase (2 u/μl) were from (Qiagen, Hilden,
Germany). The reaction was incubated at 37° C. for 90 minutes and
cooled on ice. The volume was adjusted to 6800 μl with water, yielding
a final concentration of 12.5 ng/μl of starting RNA.

[0278]For relative quantitation of the distribution of PRCP mRNA in cells
and tissues the Applied Bioscience 7900HT Sequence Detection system was
used according to the manufacturer's specifications and protocols. PCR
reactions were set up to quantitate PRCP and the housekeeping genes HPRT
(hypoxanthine phosphoribosyltransferase), GAPDH
(glyceraldehyde-3-phosphate dehydrogenase), α-actin, and others.
Forward and reverse primers and probes for PRCP were designed using the
Applied Bioscience ABI Primer Express® software and were synthesized
by Eurogentec (Belgium). The PRCP forward primer sequence was: Primer1
(SEQ ID NO: 3). The PRCP reverse primer sequence was Primer2 (SEQ ID NO:
4). Probe1 (SEQ ID NO: 5), labelled with FAM (carboxyfluorescein
succinimidyl ester) as the reporter dye and TAMRA
(carboxytetramethylrhodamine) as the quencher, is used as a probe for
PRCP. The following reagents were prepared in a total of 20 μl:
1× qPCR-MasterMix (Eurogentec; Belgium) and Probe1 (SEQ ID NO: 9),
PRCP forward and reverse primers each at 200 nM, 200 nM PRCP
FAM/TAMRA-labelled probe, and 5 μl of template cDNA. Thermal cycling
parameters were 2 mM at 50° C., followed by 10 min at 95°
C., followed by 40 cycles of melting at 95° C. for 15 sec and
annealing/extending at 60° C. for 1 min.

Calculation of Corrected CT Values

[0279]The CT (threshold cycle) value is calculated as described in the
"Quantitative determination of nucleic acids" section. The CF-value
(factor for threshold cycle correction) is calculated as follows:
[0280]1. PCR reactions were set up to quantitate the housekeeping genes
(HKG) for each cDNA sample. [0281]2. CT.sub.HKG-values (threshold cycle
for housekeeping gene) were calculated as described in the "Quantitative
determination of nucleic acids" section. [0282]3. CT.sub.HKG-mean values
(CT mean value of all HKG tested on one cDNAs) of all HKG for each cDNA
are calculated (n=number of HKG): [0283]CT.sub.HKG-n-mean
value=(CT.sub.HKG1-value+CT.sub.HKG2-value+ . . . +CT.sub.HKG-n-value)/n
[0284]4. CTpannel mean value (CT mean value of all HKG in all
tested cDNAs)=(CT.sub.HKG1-mean value+CT.sub.HKG2-mean value+ . . .
+CT.sub.HKG-y-mean value)/y (y=number of cDNAs) [0285]5. CFcDNA-n
(correction factor for cDNA n)=CTpannel-mean value-CT.sub.HKG-n-mean
value 6. CTcDNA-n (CT value of the tested gene for the cDNA
n)+CFcDNA-n (correction factor for cDNA n)=CTcor-cDNA-n
(corrected CT value for a gene on cDNA n)

[0289]Knowledge of the correct, complete cDNA sequence coding for PRCP
enables its use as a tool for antisense technology in the investigation
of gene function. Oligonucleotides, cDNA or genomic fragments comprising
the antisense strand of a polynucleotide coding for PRCP are used either
in vitro or in vivo to inhibit translation of the mRNA. Such technology
is now well known in the art, and antisense molecules can be designed at
various locations along the nucleotide sequences. By treatment of cells
or whole test animals with such antisense sequences, the gene of interest
is effectively turned off. Frequently, the function of the gene is
ascertained by observing behavior at the intracellular, cellular, tissue
or organismal level (e.g., lethality, loss of differentiated function,
changes in morphology, etc.).

[0290]In addition to using sequences constructed to interrupt
transcription of a particular open reading frame, modifications of gene
expression is obtained by designing antisense sequences to intron
regions, promoter/enhancer elements, or even to trans-acting regulatory
genes.

Example 4

Expression of PRCP

[0291]Expression of PRCP is accomplished by subcloning the cDNAs into
appropriate expression vectors and transfecting the vectors into
expression hosts such as, e.g., E. coli. In a particular case, the vector
is engineered such that it contains a promoter for β-galactosidase,
upstream of the cloning site, followed by sequence containing the
amino-terminal Methionine and the subsequent seven residues of
β-galactosidase. Immediately following these eight residues is an
engineered bacteriophage promoter useful for artificial priming and
transcription and for providing a number of unique endonuclease
restriction sites for cloning.

[0292]Induction of the isolated, transfected bacterial strain with
Isopropyl-β-D-thiogalactopyranoside (IPTG) using standard methods
produces a fusion protein corresponding to the first seven residues of
β-galactosidase, about 15 residues of "linker", and the peptide
encoded within the cDNA. Since cDNA clone inserts are generated by an
essentially random process, there is probability of 33% that the included
cDNA will lie in the correct reading frame for proper translation. If the
cDNA is not in the proper reading frame, it is obtained by deletion or
insertion of the appropriate number of bases using well known methods
including in vitro mutagenesis, digestion with exonuclease III or mung
bean nuclease, or the inclusion of an oligonucleotide linker of
appropriate length.

[0293]The PRCP cDNA is shuttled into other vectors known to be useful for
expression of proteins in specific hosts. Oligonucleotide primers
containing cloning sites as well as a segment of DNA (about 25 bases)
sufficient to hybridize to stretches at both ends of the target cDNA is
synthesized chemically by standard methods. These primers are then used
to amplify the desired gene segment by PCR. The resulting gene segment is
digested with appropriate restriction enzymes under standard conditions
and isolated by gel electrophoresis. Alternately, similar gene segments
are produced by digestion of the cDNA with appropriate restriction
enzymes. Using appropriate primers, segments of coding sequence from more
than one gene are ligated together and cloned in appropriate vectors. It
is possible to optimize expression by construction of such chimeric
sequences.

[0294]Suitable expression hosts for such chimeric molecules include, but
are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO)
and human 293 cells., insect cells such as Sf9 cells, yeast cells such as
Saccharomyces cerevisiae and bacterial cells such as E. coli. For each of
these cell systems, a useful expression vector also includes an origin of
replication to allow propagation in bacteria, and a selectable marker
such as the β-lactamase antibiotic resistance gene to allow plasmid
selection in bacteria. In addition, the vector may include a second
selectable marker such as the neomycin phosphotransferase gene to allow
selection in transfected eukaryotic host cells. Vectors for use in
eukaryotic expression hosts require RNA processing elements such as 3'
polyadenylation sequences if such are not part of the cDNA of interest.

[0295]Additionally, the vector contains promoters or enhancers which
increase gene expression. Such promoters are host specific and include
MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac
and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase
and PGH promoters for yeast. Transcription enhancers, such as the rous
sarcoma virus enhancer, are used in mammalian host cells. Once
homogeneous cultures of recombinant cells are obtained through standard
culture methods, large quantities of recombinantly produced PRCP are
recovered from the conditioned medium and analyzed using chromatographic
methods known in the art. For example, PRCP can be cloned into the
expression vector pcDNA3, as exemplified herein. This product can be used
to transform, for example, HEK293 or COS by methodology standard in the
art. Specifically, for example, using Lipofectamine (Gibco BRL catolog
no. 18324-020) mediated gene transfer.

Example 5

Isolation of Recombinant PRCP

[0296]PRCP is expressed as a chimeric protein with one or more additional
polypeptide domains added to facilitate protein purification. Such
purification facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals [Appa Rao, 1997] and the domain
utilized in the FLAGS extension/affinity purification system (Immunex
Corp., Seattle, Wash.). The inclusion of a cleavable linker sequence such
as Factor Xa or enterokinase (Invitrogen, Groningen, The Netherlands)
between the purification domain and the PRCP sequence is useful to
facilitate expression of PRCP.

[0299]cDNA encoding proteases cloned into either the donor plasmid
pFASTBAC1 or pFASTBAC-HT which contain a mini-Tn7 transposition element.
The recombinant plasmid is transformed into DH10BAC competent cells which
contain the parent bacmid bMON14272 (AcNPV infectious DNA) and a helper
plasmid. The mini-Tn7 element on the pFASTBAC donor can transpose to the
attTn7 attachment site on the bacmid thus introducing the protease gene
into the viral genome. Colonies containing recombinant bacmids are
identified by disruption of the lacZ gene. The protease/bacmid construct
can then be isolated and infected into insect cells (Sf9 cells) resulting
in the production of infectious recombinant baculovirus particles and
expression of either unfused recombinant enzyme (pFastbac1) or PRCP-His
fusion protein (pFastbacHT).

[0300]Cells are harvested and extracts prepared 24, 48 and 72 hours after
transfection. Expression of PRCP is confirmed by coomassie staining after
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
western blotting onto a PVDF membrane of an unstained SDS-PAGE. The
protease-His fusion protein is detected due to the interaction between
the Ni-NTA HRP conjugate and the His-tag which is fused to PRCP.

Example 6

Production of PRCP Specific Antibodies

[0301]Two approaches are utilized to raise antibodies to PRCP, and each
approach is useful for generating either polyclonal or monoclonal
antibodies. In one approach, denatured protein from reverse phase HPLC
separation is obtained in quantities up to 75 mg. This denatured protein
is used to immunize mice or rabbits using standard protocols; about 100
μg are adequate for immunization of a mouse, while up to 1 mg might be
used to immunize a rabbit. For identifying mouse hybridomas, the
denatured protein is radioiodinated and used to screen potential murine
B-cell hybridomas for those which produce antibody. This procedure
requires only small quantities of protein, such that 20 mg is sufficient
for labeling and screening of several thousand clones.

[0302]In the second approach, the amino acid sequence of an appropriate
PRCP domain, as deduced from translation of the cDNA, is analyzed to
determine regions of high antigenicity. Oligopeptides comprising
appropriate hydrophilic regions are synthesized and used in suitable
immunization protocols to raise antibodies. The optimal amino acid
sequences for immunization are usually at the C-terminus, the N-terminus
and those intervening, hydrophilic regions of the polypeptide which are
likely to be exposed to the external environment when the protein is in
its natural conformation.

[0303]Typically, selected peptides, about 15 residues in length, are
synthesized using an Applied Biosystems Peptide Synthesizer Model 431A
using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH;
Sigma, St. Louis, Mo.) by reaction with
M-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS. If necessary, a
cysteine is introduced at the N-terminus of the peptide to permit
coupling to KLH. Rabbits are immunized with the peptide-KLH complex in
complete Freund's adjuvant. The resulting antisera are tested for
antipeptide activity by binding the peptide to plastic, blocking with 1%
bovine serum albumin, reacting with antisera, washing and reacting with
labeled (radioactive or fluorescent), affinity purified, specific goat
anti-rabbit IgG.

[0304]Hybridomas are prepared and screened using standard techniques.
Hybridomas of interest are detected by screening with labeled PRCP to
identify those fusions producing the monoclonal antibody with the desired
specificity. In a typical protocol, wells of plates (FAST;
Becton-Dickinson, Palo Alto, Calif.) are coated during incubation with
affinity purified, specific rabbit anti-mouse (or suitable antispecies 1
g) antibodies at 10 mg/ml. The coated wells are blocked with 1% bovine
serum albumin, (BSA), washed and incubated with supernatants from
hybridomas. After washing the wells are incubated with labeled PRCP at 1
mg/ml. Supernatants with specific antibodies bind more labeled PRCP than
is detectable in the background. Then clones producing specific
antibodies are expanded and subjected to two cycles of cloning at
limiting dilution. Cloned hybridomas are injected into pristane-treated
mice to produce ascites, and monoclonal antibody is purified from mouse
ascitic fluid by affinity chromatography on Protein A. Monoclonal
antibodies with affinities of at least

[0305]108 M-1, preferably 109 to 1010 M-1 or
stronger, are typically made by standard procedures.

Example 7

Diagnostic Test Using PRCP Specific Antibodies

[0306]Particular PRCP antibodies are useful for investigating signal
transduction and the diagnosis of infectious or hereditary conditions
which are characterized by differences in the amount or distribution of
PRCP or downstream products of an active signaling cascade.

[0307]Diagnostic tests for PRCP include methods utilizing antibody and a
label to detect PRCP in human body fluids, membranes, cells, tissues or
extracts of such. The polypeptides and antibodies of the present
invention are used with or without modification. Frequently, the
polypeptides and antibodies are labeled by joining them, either
covalently or noncovalently, with a substance which provides for a
detectable signal. A wide variety of labels and conjugation techniques
are known and have been reported extensively in both the scientific and
patent literature. Suitable labels include radionuclides, enzymes,
substrates, cofactors, inhibitors, fluorescent agents, chemilumines-cent
agents, chromogenic agents, magnetic particles and the like.

[0308]A variety of protocols for measuring soluble or membrane-bound PRCP,
using either polyclonal or monoclonal antibodies specific for the
protein, are known in the art. Examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent
activated cell sorting (FACS). A two-site monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering epitopes
on PRCP is preferred, but a competitive binding assay may be employed.

Example 8

Purification of Native PRCP Using Specific Antibodies

[0309]Native or recombinant PRCP is purified by immunoaffinity
chromatography using antibodies specific for PRCP. In general, an
immunoaffinity column is constructed by covalently coupling the anti-TRH
antibody to an activated chromatographic resin.

[0310]Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium sulfate or by purification on immobilized
Protein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise,
monoclonal antibodies are prepared from mouse ascites fluid by ammonium
sulfate precipitation or chromatography on immobilized Protein A.
Partially purified immunoglobulin is covalently attached to a
chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB
Biotechnology). The antibody is coupled to the resin, the resin is
blocked, and the derivative resin is washed according to the
manufacturer's instructions.

[0311]Such immunoaffinity columns are utilized in the purification of PRCP
by preparing a fraction from cells containing PRCP in a soluble form.
This preparation is derived by solubilization of whole cells or of a
subcellular fraction obtained via differential centrifugation (with or
without addition of detergent) or by other methods well known in the art.
Alternatively, soluble PRCP containing a signal sequence is secreted in
useful quantity into the medium in which the cells are grown.

[0312]A soluble PRCP-containing preparation is passed over the
immunoaffinity column, and the column is washed under conditions that
allow the preferential absorbance of PRCP (e.g., high ionic strength
buffers in the presence of detergent). Then, the column is eluted under
conditions that disrupt antibody/protein binding (e.g., a buffer of pH
2-3 or a high concentration of a chaotrope such as urea or thiocyanate
ion), and PRCP is collected.

Example 9

Drug Screening

[0313]This invention is particularly useful for screening therapeutic
compounds by using PRCP or fragments thereof in any of a variety of drug
screening techniques.

[0314]The following example provides a system for drug screening measuring
the protease activity.

[0315]The recombinant protease-His fusion protein can be purified from the
crude lysate by metal-affinity chromatography using Ni-NTA agarose. This
allows the specific retention of the recombinant material (since this is
fused to the His-tag) whilst the endogenous insect proteins are washed
off. The recombinant material is then eluted by competition with
imidazol.

[0316]The activity of PRCP molecules of the present invention can be
measured using a variety of assays that measure PRCP activity. For
example, PRCP enzyme activity can be assessed by a standard in vitro
serine/metallo/ . . . protease assay (see, for example, [U.S. Pat. No.
5,057,414]). Those of skill in the art are aware of a variety of
substrates suitable for in vitro assays, such as SucAla-Ala-Pro-Phe-pNA,
fluorescein mono-p-guanidinobenzoate hydrochloride,
benzyloxycarbonyl-L-Arginyl-5-benzyl-ester, Nalpha-Benzoyl-L-arginine
ethyl ester hydrochloride, and the like. In addition, protease assay kits
available from commercial sources, such as Calbiochem® (San Diego,
Calif.). For general references, see Barrett (Ed.), Methods in
Enzymology, Proteolytic Enzymes: Serine and Cysteine Peptidases (Academic
Press Inc. 1994), and Barrett et al., (Eds.), Handbook of Proteolytic
Enzymes (Academic Press Inc. 1998).

Example 10

Rational Drug Design

[0317]The goal of rational drug design is to produce structural analogs of
biologically active polypeptides of interest or of small molecules with
which they interact, agonists, antagonists, or inhibitors. Any of these
examples are used to fashion drugs which are more active or stable forms
of the polypeptide or which enhance or interfere with the function of a
polypeptide in vivo.

[0318]In one approach, the three-dimensional structure of a protein of
interest, or of a protein-inhibitor complex, is determined by x-ray
crystallography, by computer modeling or, most typically, by a
combination of the two approaches. Both the shape and charges of the
polypeptide must be ascertained to elucidate the structure and to
determine active site(s) of the molecule. Less often, useful information
regarding the structure of a polypeptide is gained by modeling based on
the structure of homologous proteins. In both cases, relevant structural
information is used to design efficient inhibitors. Useful examples of
rational drug design include molecules which have improved activity or
stability or which act as inhibitors, agonists, or antagonists of native
peptides.

[0319]It is also possible to isolate a target-specific antibody, selected
by functional assay, as described above, and then to solve its crystal
structure. This approach, in principle, yields a pharmacore upon which
subsequent drug design is based. It is possible to bypass protein
crystallography altogether by generating anti-idiotypic antibodies
(anti-ids) to a functional, pharmacologically active antibody. As a
mirror image of a mirror image, the binding site of the anti-ids is
expected to be an analog of the original receptor. The anti-id is then
used to identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides then act as the
pharmacore.

[0320]By virtue of the present invention, sufficient amount of polypeptide
are made available to perform such analytical studies as X-ray
crystallography. In addition, knowledge of the PRCP amino acid sequence
provided herein provides guidance to those employing computer modeling
techniques in place of or in addition to x-ray crystallography.

Example 11

Identification of Other Members of the Signal Transduction Complex

[0321]Labeled PRCP is useful as a reagent for the purification of
molecules with which it interacts. In one embodiment of affinity
purification, PRCP is covalently coupled to a chromatography column.
Cell-free extract derived from synovial cells or putative target cells is
passed over the column, and molecules with appropriate affinity bind to
PRCP. PRCP-complex is recovered from the column, and the PRCP-binding
ligand disassociated and subjected to N-terminal protein sequencing. The
amino acid sequence information is then used to identify the captured
molecule or to design degenerate oligonucleotide probes for cloning the
relevant gene from an appropriate cDNA library.

[0322]In an alternate method, antibodies are raised against PRCP,
specifically monoclonal antibodies. The monoclonal antibodies are
screened to identify those which inhibit the binding of labeled PRCP.
These monoclonal antibodies are then used therapeutically.

Example 12

Use and Administration of Antibodies, Inhibitors, or Antagonists

[0323]Antibodies, inhibitors, or antagonists of PRCP or other treatments
and compounds that are limiters of signal transduction (LSTs), provide
different effects when administered therapeutically. LSTs are formulated
in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium
preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH
may vary according to the characteristics of the antibody, inhibitor, or
antagonist being formulated and the condition to be treated.
Characteristics of LSTs include solubility of the molecule, its half-life
and antigenicity/immunogenicity. These and other characteristics aid in
defining an effective carrier. Native human proteins are preferred as
LSTs, but organic or synthetic molecules resulting from drug screens are
equally effective in particular situations.

[0324]LSTs are delivered by known routes of administration including but
not limited to topical creams and gels; transmucosal spray and aerosol;
transdermal patch and bandage; injectable, intravenous and lavage
formulations; and orally administered liquids and pills particularly
formulated to resist stomach acid and enzymes. The particular
formulation, exact dosage, and route of administration is determined by
the attending physician and varies according to each specific situation.

[0325]Such determinations are made by considering multiple variables such
as the condition to be treated, the LST to be administered, and the
pharmacokinetic profile of a particular LST. Additional factors which are
taken into account include severity of the disease state, patient's age,
weight, gender and diet, time and frequency of LST administration,
possible combination with other drugs, reaction sensitivities, and
tolerance/response to therapy. Long acting LST formulations might be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular LST.

[0326]Normal dosage amounts vary from 0.1 to 105 μg, up to a total
dose of about 1 g, depending upon the route of administration. Guidance
as to particular dosages and methods of delivery is provided in the
literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. Those
skilled in the art employ different formulations for different LSTs.
Administration to cells such as nerve cells necessitates delivery in a
manner different from that to other cells such as vascular endothelial
cells.

[0327]It is contemplated that abnormal signal transduction, trauma, or
diseases which trigger PRCP activity are treatable with LSTs. These
conditions or diseases are specifically diagnosed by the tests discussed
above, and such testing should be performed in suspected cases of viral,
bacterial or fungal infections, allergic responses, mechanical injury
associated with trauma, hereditary diseases, lymphoma or carcinoma, or
other conditions which activate the genes of lymphoid or neuronal
tissues.

Example 13

Production of Non-human Transgenic Animals

[0328]Animal model systems which elucidate the physiological and
behavioral roles of the PRCP are produced by creating nonhuman transgenic
animals in which the activity of the PRCP is either increased or
decreased, or the amino acid sequence of the expressed PRCP is altered,
by a variety of techniques. Examples of these techniques include, but are
not limited to: 1) Insertion of normal or mutant versions of DNA encoding
a PRCP, by microinjection, electroporation, retroviral transfection or
other means well known to those skilled in the art, into appropriately
fertilized embryos in order to produce a transgenic animal or 2)
homologous recombination of mutant or normal, human or animal versions of
these genes with the native gene locus in transgenic animals to alter the
regulation of expression or the structure of these PRCP sequences. The
technique of homologous recombination is well known in the art. It
replaces the native gene with the inserted gene and hence is useful for
producing an animal that cannot express native PRCPs but does express,
for example, an inserted mutant PRCP, which has replaced the native PRCP
in the animal's genome by recombination, resulting in underexpression of
the transporter. Microinjection adds genes to the genome, but does not
remove them, and the technique is useful for producing an animal which
expresses its own and added PRCP, resulting in overexpression of the
PRCP.

[0329]One means available for producing a transgenic animal, with a mouse
as an example, is as follows: Female mice are mated, and the resulting
fertilized eggs are dissected out of their oviducts. The eggs are stored
in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA
encoding PRCP is purified from a vector by methods well known to the one
skilled in the art. Inducible promoters may be fused with the coding
region of the DNA to provide an experimental means to regulate expression
of the transgene. Alternatively or in addition, tissue specific
regulatory elements may be fused with the coding region to permit
tissue-specific expression of the transgene. The DNA, in an appropriately
buffered solution, is put into a microinjection needle (which may be made
from capillary tubing using a piper puller) and the egg to be injected is
put in a depression slide. The needle is inserted into the pronucleus of
the egg, and the DNA solution is injected. The injected egg is then
transferred into the oviduct of a pseudopregnant mouse which is a mouse
stimulated by the appropriate hormones in order to maintain false
pregnancy, where it proceeds to the uterus, implants, and develops to
term. As noted above, microinjection is not the only method for inserting
DNA into the egg but is used here only for exemplary purposes.